RESUMEN
Idiopathic pulmonary fibrosis (IPF) is characterized by an aberrant repair response with uncontrolled turnover of extracellular matrix involving mesenchymal cell phenotypes, where lung resident mesenchymal stem cells (LRMSC) have been supposed to have an important role. However, the contribution of LRMSC in lung fibrosis is not fully understood, and the role of LRMSC in IPF remains to be elucidated. Here, we performed transcriptomic and functional analyses on LRMSC isolated from IPF and control patients (CON). Both over-representation and gene set enrichment analyses indicated that oxidative phosphorylation is the major dysregulated pathway in IPF LRMSC. The most relevant differences in biological processes included complement activation, mesenchyme development, and aerobic electron transport chain. Compared to CON LRMSC, IPF cells displayed impaired mitochondrial respiration, lower expression of genes involved in mitochondrial dynamics, and dysmorphic mitochondria. These changes were linked to an impaired autophagic response and a lower mRNA expression of pro-apoptotic genes. In addition, IPF TGFß-exposed LRMSC presented different expression profiles of mitochondrial-related genes compared to CON TGFß-treated cells, suggesting that TGFß reinforces mitochondrial dysfunction. In conclusion, these results suggest that mitochondrial dysfunction is a major event in LRMSC and that their occurrence might limit LRMSC function, thereby contributing to IPF development.
Asunto(s)
Fibrosis Pulmonar Idiopática , Células Madre Mesenquimatosas , Humanos , Fibrosis Pulmonar Idiopática/genética , Autofagia , Mitocondrias , PulmónRESUMEN
Many members of the forkhead genes family of transcription factors have been implicated as important regulators of metabolism, in particular, glucose homeostasis, e.g., Foxo1, Foxa3, and Foxc2. The purpose of this study was to exploit the possibility that yet unknown members of this gene family play a role in regulating glucose tolerance in adipocytes. We identified Foxf2 in a screen for adipose-expressed forkhead genes. In vivo overexpression of Foxf2 in an adipose tissue-restricted fashion demonstrated that such mice display a significantly induced insulin secretion in response to an intravenous glucose load compared with wild-type littermates. In response to increased Foxf2 expression, insulin receptor substrate 1 (IRS1) mRNA and protein levels are significantly downregulated in adipocytes; however, the ratio of serine vs. tyrosine phosphorylation of IRS1 seems to remain unaffected. Furthermore, adipocytes overexpressing Foxf2 have a significantly lower insulin-mediated glucose uptake compared with wild-type adipocytes. These findings argue that Foxf2 is a previously unrecognized regulator of cellular and systemic whole body glucose tolerance, at least in part, due to lower levels of IRS1. Foxf2 and its downstream target genes can provide new insights with regard to identification of novel therapeutic targets.
Asunto(s)
Tejido Adiposo/metabolismo , Factores de Transcripción Forkhead/metabolismo , Glucosa/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Células 3T3 , Adipocitos , Animales , Regulación hacia Abajo , Expresión Génica , Homeostasis/genética , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
We have developed a generic model for in vitro high-throughput screening for agents regulating transcription of genes in the mouse genome here exemplified by Foxc2, a forkhead transcription factor involved in regulation of adipocyte metabolism. We made a Foxc2-LacZ reporter "knock-in" mouse in which one of the two Foxc2 alleles has been inactivated and replaced by a LacZ reporter gene. Mouse embryonic fibroblasts, derived from such mice, were differentiated in vitro to adipocytes and used in cell-based screens. Forskolin as well as 12-O-tetradecanoylphorbol-13-acetate (TPA) increased levels of Foxc2nLacZ fusion protein. We could also demonstrate that this was paralleled by an increase in Foxc2 mRNA, transcribed from the wild type allele. This generic method offers a novel way of identifying both positive and negative upstream regulators of a gene, using high-throughput screening methodology. In a cell-based screen using such methodology we demonstrate efficacy by identifying NKH477 as a Foxc2 activating compound.
Asunto(s)
Adipocitos/citología , Diferenciación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Factores de Transcripción Forkhead/genética , Bibliotecas de Moléculas Pequeñas , Adipocitos/metabolismo , Animales , Células Cultivadas , Embrión de Mamíferos/citología , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Técnicas de Sustitución del Gen , Operón Lac , Masculino , RatonesRESUMEN
In this study, we explore the effects of several FOX and mutant FOX transcription factors on adipocyte determination, differentiation, and metabolism. In addition to Foxc2 and Foxo1, we report that Foxf2, Foxp1, and Foxa1 are other members of the Fox family that show regulated expression during adipogenesis. Although enforced expression of FOXC2 inhibits adipogenesis, Foxf2 slightly enhances the rate of differentiation. Constitutively active FOXC2-VP16 inhibits adipogenesis through multiple mechanisms. FOXC2-VP16 impairs the transient induction of C/EBPbeta during adipogenesis and induces expression of the transcriptional repressor Hey1 as well as the activator of Wnt/beta-catenin signaling, Wnt10b. The constitutive transcriptional repressor, FOXC2-Eng, enhances adipogenesis of preadipocytes and multipotent mesenchymal precursors and determines NIH-3T3 and C2C12 cells to the adipocyte lineage. Although PPARgamma ligand or C/EBPalpha are not necessary for stimulation of adipogenesis by FOXC2-Eng, at least low levels of PPARgamma protein are absolutely required. Finally, expression of FOXC2-Eng in adipocytes increases insulin-stimulated glucose uptake, further expanding the profound and pleiotropic effects of FOX transcription factors on adipocyte biology.
Asunto(s)
Adipocitos/fisiología , Adipogénesis/fisiología , Diferenciación Celular/fisiología , Factores de Transcripción Forkhead/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Células 3T3-L1 , Adipocitos/citología , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica , Humanos , Ratones , PPAR gamma/antagonistas & inhibidores , PPAR gamma/genética , PPAR gamma/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Insulin resistance is a common disorder caused by a wide variety of physiological insults, some of which include poor diet, inflammation, anti-inflammatory steroids, hyperinsulinemia, and dyslipidemia. The common link between these diverse insults and insulin resistance is widely considered to involve impaired insulin signaling, particularly at the level of the insulin receptor substrate (IRS). To test this model, we utilized a heterologous system involving the platelet-derived growth factor (PDGF) pathway that recapitulates many aspects of insulin action independently of IRS. We comprehensively analyzed six models of insulin resistance in three experimental systems and consistently observed defects in both insulin and PDGF action despite a range of insult-specific defects within the IRS-Akt nexus. These findings indicate that while insulin resistance is associated with multiple deficiencies, the most deleterious defects and the origin of insulin resistance occur independently of IRS.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Hiperinsulinismo/etiología , Hipoglucemiantes/farmacología , Resistencia a la Insulina , Insulina/farmacología , Adipocitos/citología , Adipocitos/metabolismo , Animales , Células Cultivadas , Prueba de Tolerancia a la Glucosa , Transportador de Glucosa de Tipo 4/metabolismo , Immunoblotting , Inflamación , Proteínas Sustrato del Receptor de Insulina , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mioblastos/citología , Mioblastos/metabolismo , Estrés Oxidativo , Palmitatos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Transducción de SeñalRESUMEN
Hyperglycemia is a defining feature of Type 1 and 2 diabetes. Hyperglycemia also causes insulin resistance, and our group (Kraegen EW, Saha AK, Preston E, Wilks D, Hoy AJ, Cooney GJ, Ruderman NB. Am J Physiol Endocrinol Metab Endocrinol Metab 290: E471-E479, 2006) has recently demonstrated that hyperglycemia generated by glucose infusion results in insulin resistance after 5 h but not after 3 h. The aim of this study was to investigate possible mechanism(s) by which glucose infusion causes insulin resistance in skeletal muscle and in particular to examine whether this was associated with changes in insulin signaling. Hyperglycemia (~10 mM) was produced in cannulated male Wistar rats for up to 5 h. The glucose infusion rate required to maintain this hyperglycemia progressively lessened over 5 h (by 25%, P < 0.0001 at 5 h) without any alteration in plasma insulin levels consistent with the development of insulin resistance. Muscle glucose uptake in vivo (44%; P < 0.05) and glycogen synthesis rate (52%; P < 0.001) were reduced after 5 h compared with after 3 h of infusion. Despite these changes, there was no decrease in the phosphorylation state of multiple insulin signaling intermediates [insulin receptor, Akt, AS160 (Akt substrate of 160 kDa), glycogen synthase kinase-3beta] over the same time course. In isolated soleus strips taken from control or 1- or 5-h glucose-infused animals, insulin-stimulated 2-deoxyglucose transport was similar, but glycogen synthesis was significantly reduced in the 5-h muscle sample (68% vs. 1-h sample; P < 0.001). These results suggest that the reduced muscle glucose uptake in rats after 5 h of acute hyperglycemia is due more to the metabolic effects of excess glycogen storage than to a defect in insulin signaling or glucose transport.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Glucosa/administración & dosificación , Hiperglucemia/metabolismo , Resistencia a la Insulina/fisiología , Músculo Esquelético/metabolismo , Proteína Oncogénica v-akt/metabolismo , Animales , Glucemia/metabolismo , Glucosa/metabolismo , Glucógeno/metabolismo , Técnicas In Vitro , Infusiones Intravenosas , Insulina/sangre , Masculino , Fosforilación , Distribución Aleatoria , Ratas , Ratas Wistar , Transducción de SeñalRESUMEN
Overexpression of forkhead transcription factor FOXC2 in white adipose tissue (WAT) leads to a lean phenotype resistant to diet-induced obesity. This is due, in part, to enhanced catecholamine-induced cAMP-PKA signaling in FOXC2 transgenic mice. Here we show that rolipram treatment of adipocytes from FOXC2 transgenic mice did not increase isoproterenol-induced cAMP accumulation to the same extent as in wild type cells. Accordingly, phosphodiesterase-4 (PDE4) activity was reduced by 75% and PDE4A5 protein expression reduced by 30-50% in FOXC2 transgenic WAT compared to wild type. Thus, reduced PDE4 activity in adipocytes from FOXC2 transgenic mice contributes to amplified beta-AR induced cAMP responses observed in these cells.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , AMP Cíclico/biosíntesis , Factores de Transcripción Forkhead/biosíntesis , Transducción de Señal/fisiología , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Adipocitos/citología , Tejido Adiposo/citología , Agonistas Adrenérgicos beta/farmacología , Animales , Catecolaminas/farmacología , Células Cultivadas , AMP Cíclico/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Factores de Transcripción Forkhead/genética , Expresión Génica/genética , Isoproterenol/farmacología , Ratones , Ratones Transgénicos , Obesidad/genética , Obesidad/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Receptores Adrenérgicos beta/metabolismo , Rolipram/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
To gain insight into the expression pattern and functional importance of the forkhead transcription factor Foxs1, we constructed a Foxs1-beta-galactosidase reporter gene "knock-in" (Foxs1beta-gal/beta-gal) mouse, in which the wild-type (wt) Foxs1 allele has been inactivated and replaced by a beta-galactosidase reporter gene. Staining for beta-galactosidase activity reveals an expression pattern encompassing neural crest-derived cells, e.g., cranial and dorsal root ganglia as well as several other cell populations in the central nervous system (CNS), most prominently the internal granule layer of cerebellum. Other sites of expression include the lachrymal gland, outer nuclear layer of retina, enteric ganglion neurons, and a subset of thalamic and hypothalamic nuclei. In the CNS, blood vessel-associated smooth muscle cells and pericytes stain positive for Foxs1. Foxs1beta-gal/beta-gal mice perform significantly better (P < 0.01) on a rotating rod than do wt littermates. We have also noted a lower body weight gain (P < 0.05) in Foxs1beta-gal/lbeta-gal males on a high-fat diet, and we speculate that dorsomedial hypothalamic neurons, expressing Foxs1, could play a role in regulating body weight via regulation of sympathetic outflow. In support of this, we observed increased levels of uncoupling protein 1 mRNA in Foxs1beta-gal/beta-gal mice. This points toward a role for Foxs1 in the integration and processing of neuronal signals of importance for energy turnover and motor function.
Asunto(s)
Peso Corporal/genética , Sistema Nervioso Central/crecimiento & desarrollo , Sistema Nervioso Central/metabolismo , Actividad Motora/genética , Cresta Neural/metabolismo , Factores de Transcripción/genética , Animales , Peso Corporal/fisiología , Sistema Nervioso Central/citología , Sistema Nervioso Central/embriología , Factores de Transcripción Forkhead , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Inmunohistoquímica , Ratones , Ratones Mutantes , Actividad Motora/fisiología , Cresta Neural/citología , Cresta Neural/embriología , Cresta Neural/crecimiento & desarrollo , Reacción en Cadena de la Polimerasa , Rotación , Análisis de Secuencia de ADN , Factores de Tiempo , Distribución Tisular , Factores de Transcripción/metabolismo , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Insulin resistance plays a major role in the development of type 2 diabetes and may be causally associated with increased intracellular fat content. Transgenic mice with adipocyte-specific overexpression of FOXC2 (forkhead transcription factor) have been generated and shown to be protected against diet-induced obesity and glucose intolerance. To understand the underlying mechanism, we examined the effects of chronic high-fat feeding on tissue-specific insulin action and glucose metabolism in the FOXC2 transgenic (Tg) mice. Whole-body fat mass were significantly reduced in the FOXC2 Tg mice fed normal diet or high-fat diet compared with the wild-type mice. Diet-induced insulin resistance in skeletal muscle of the wild-type mice was associated with defects in insulin signaling and significant increases in intramuscular fatty acyl CoA levels. In contrast, FOXC2 Tg mice were completely protected from diet-induced insulin resistance and intramuscular accumulation of fatty acyl CoA. High-fat feeding also blunted insulin-mediated suppression of hepatic glucose production in the wild-type mice, whereas FOXC2 Tg mice were protected from diet-induced hepatic insulin resistance. These findings demonstrate an important role of adipocyte-expressed FOXC2 on whole-body glucose metabolism and further suggest FOXC2 as a novel therapeutic target for the treatment of insulin resistance and type 2 diabetes.
Asunto(s)
Acilcoenzima A/metabolismo , Adipocitos/metabolismo , Proteínas de Unión al ADN/fisiología , Grasas de la Dieta/metabolismo , Resistencia a la Insulina , Músculo Esquelético/metabolismo , Factores de Transcripción/fisiología , Animales , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Factores de Transcripción Forkhead , Expresión Génica , Insulina/metabolismo , Masculino , Ratones , Ratones Transgénicos , Transducción de Señal , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
The boundary cap (BC) is a transient neural crest-derived group of cells located at the dorsal root entry zone (DREZ) that have been shown to differentiate into sensory neurons and glia in vivo. We find that when placed in culture, BC cells self-renew, show multipotency in clonal cultures and express neural crest stem cell (NCSCs) markers. Unlike sciatic nerve NCSCs, the BC-NCSC (bNCSCs) generates sensory neurons upon differentiation. The bNCSCs constitute a common source of cells for functionally diverse types of neurons, as a single bNCSC can give rise to several types of nociceptive and thermoreceptive sensory neurons. Our data suggests that BC cells comprise a source of multipotent sensory specified stem cells that persist throughout embryogenesis.
Asunto(s)
Diferenciación Celular/fisiología , Ganglios Espinales/embriología , Células Madre Multipotentes/citología , Cresta Neural/citología , Neuronas Aferentes/citología , Animales , Calcio/metabolismo , Células Cultivadas , Cartilla de ADN , Fluorometría , Galactósidos , Inmunohistoquímica , Hibridación in Situ , Indoles , Ratones , Ratones Transgénicos , Células Madre Multipotentes/metabolismo , Células Madre Multipotentes/fisiología , Cresta Neural/metabolismo , Cresta Neural/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
As a result of selecting triglycerides as the major vehicle for storing superfluous energy, evolution came up with a specialized cell type, the adipocyte, equipped to handle triglycerides and its potentially toxic metabolites--fatty acids. For the first time in history large human populations are subjected a wealth of cheap, accessible and palatable calories. This has created a situation, on a large scale not previously encountered, in which the capacity to store triglycerides in adipocytes is an important determinant of human health. Too few adipocytes (e.g. lipodystrophia) or a situation in which all adipocytes are filled, to their maximum capacity (e.g. severe obesity), will create very similar and unfavorable metabolic situations in which ectopic triglyceride stores will appear in tissues like liver and muscle. This review sets out to discuss the adipocyte and its role in metabolism as well as the consequences of a metabolic situation, in which the adipocyte has lost its fat storing monopoly.
Asunto(s)
Diabetes Mellitus Tipo 2 , Resistencia a la Insulina , Tejido Adiposo/fisiología , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Obesidad/metabolismoRESUMEN
The human transcription factor FOXC2 has recently been shown to protect against diet-induced insulin resistance in transgenic mice. We investigated the expression of FOXC2 in fat and muscle and performed a genetic analysis in human subjects. FOXC2 mRNA levels were increased in visceral compared with subcutaneous fat from obese subjects (12 +/- 4-fold; P = 0.0001), and there was a correlation between whole-body insulin sensitivity and FOXC2 mRNA levels in visceral fat (fS-insulin R = -0.64, P = 0.01, and homeostasis model assessment of insulin resistance [HOMA-IR] R = -0.68, P = 0.007) and skeletal muscle (fS-insulin R = -0.57, P = 0.03, and HOMA-IR R = -0.55, P = 0.04). Mutation screening of the FOXC2 gene identified a common polymorphism in the 5' untranslated region (C-512T). The T allele was associated with enhanced insulin sensitivity (HOMA-IR P = 0.007) and lower plasma triglyceride levels in females (P = 0.007). Also, the higher expression of FOXC2 in visceral than in subcutaneous fat was restricted to subjects homozygous for the T allele (P = 0.03 vs. P = 0.7). Our data suggest that increased FOXC2 expression may protect against insulin resistance in human subjects and that genetic variability in the gene may influence features associated with the metabolic syndrome.
Asunto(s)
Regiones no Traducidas 5'/genética , Proteínas de Unión al ADN/genética , Resistencia a la Insulina/fisiología , ARN Mensajero/metabolismo , Factores de Transcripción/genética , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Adulto , Células Cultivadas , Femenino , Factores de Transcripción Forkhead , Humanos , Insulina/farmacología , Resistencia a la Insulina/genética , Masculino , Persona de Mediana Edad , Músculo Esquelético/metabolismo , Polimorfismo Genético/fisiología , Tejido Subcutáneo/metabolismo , Regulación hacia Arriba , Vísceras/metabolismoRESUMEN
We have reported recently that mice overexpressing the forkhead/winged helix transcription factor FOXC2 are lean and show increased responsiveness to insulin due to sensitization of the beta-adrenergic cAMP-PKA(+) pathway and increased levels of the RI alpha subunit of cAMP-dependent protein kinase (PKA) (Cederberg, A., Grønning, L. M., Ahren, B., Taskén, K., Carlsson, P., and Enerbäck, S. (2001) Cell 106, 563-573). In this present study, we reveal that FOXC2 and a related factor, FOXD1, specifically activate the 1b promoter of the RI alpha gene in adipocytes and testicular Sertoli cells, respectively. By deletional mapping, we discovered two different mechanisms by which the Fox proteins activated expression from the RI alpha 1b promoter. In 3T3-L1 adipocytes, an upstream region represses promoter activity under basal conditions. Bandshift experiments indicate that overexpression of FOXC2 promotes the release of a potential repressor from this region. In Sertoli cells, sequences downstream of the transcription start sites mediate the activating effect of FOXD1, and protein kinase B alpha/Akt1 strongly induces this effect. Furthermore, we show that an inactive FOXD1 mutant lowers the cAMP-mediated induction of the RI alpha 1b reporter construct. In summary, winged helix transcription factors of the FOXC/FOXD families function as regulators of the RI alpha subunit of PKA and may integrate hormonal signals acting through protein kinase B and cAMP in a cell-specific manner.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Enzimológica de la Expresión Génica , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas , Factores de Transcripción/metabolismo , Células 3T3 , Animales , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/química , Activación Enzimática , Factores de Transcripción Forkhead , Genes Reporteros , Immunoblotting , Luciferasas/metabolismo , Masculino , Ratones , Microscopía Fluorescente , Modelos Biológicos , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-akt , Ratas , Ratas Sprague-Dawley , Células de Sertoli/metabolismo , Transducción de Señal , TransfecciónRESUMEN
We have recently identified the winged helix/forkhead gene Foxc2 as a key regulator of adipocyte metabolism that counteracts obesity and diet-induced insulin resistance. This study was performed to elucidate the hormonal regulation of Foxc2 in adipocytes. We find that TNF alpha and insulin induce Foxc2 mRNA in differentiated 3T3-L1 cells with the kinetics of an immediate early response (1-2 h with 100 ng/ml insulin or 5 ng/ml TNF alpha). This induction is, in both cases, attenuated by the PI3K inhibitor wortmannin as well as the MAPK kinase inhibitor PD98059. Furthermore, we show that stimulation of 3T3-L1 adipocytes with phorbol-12-myristate-13-acetate or 8-(4-chlorophenyl)thio-cAMP induces the expression of Foxc2. Interestingly, we find that the basal level of Foxc2 mRNA is down-regulated whereas hormonal responsiveness increases during differentiation of 3T3-L1 from preadipocytes to adipocytes. At the protein level, immunoblots with Foxc2 antibody demonstrated an induction of Foxc2 by insulin and TNF alpha in nuclear extracts of 3T3-L1 adipocytes. EMSA of nuclear proteins from phorbol-12-myristate-13-acetate- and TNF alpha-treated 3T3-L1 adipocytes using a forkhead consensus oligonucleotide revealed specific binding of a Foxc2/DNA complex. In conclusion, our data suggest that insulin and TNF alpha regulate the expression of Foxc2 via a PI3K- and ERK 1/2-dependent pathway in 3T3-L1 adipocytes. Also, signaling pathways downstream of PKA and PKC induce the expression of Foxc2 mRNA.