RESUMEN
AIMS/HYPOTHESIS: Type 2 diabetes is characterised by progressive loss of pancreatic beta cell mass and function. Therefore, it is of therapeutic interest to identify factors with the potential to improve beta cell proliferation and insulin secretion. Bone morphogenetic protein 4 (BMP4) expression is increased in diabetic animals and BMP4 reduces glucose-stimulated insulin secretion (GSIS). Here, we investigate the molecular mechanism behind this inhibition. METHODS: BMP4-mediated inhibition of GSIS was investigated in detail using single cell electrophysiological measurements and live cell Ca(2+) imaging. BMP4-mediated gene expression changes were investigated by microarray profiling, quantitative PCR and western blotting. RESULTS: Prolonged exposure to BMP4 reduced GSIS from rodent pancreatic islets. This inhibition was associated with decreased exocytosis due to a reduced Ca(2+) current through voltage-dependent Ca(2+) channels. To identify proteins involved in the inhibition of GSIS, we investigated global gene expression changes induced by BMP4 in neonatal rat pancreatic islets. Expression of the Ca(2+)-binding protein calbindin1 was significantly induced by BMP4. Overexpression of calbindin1 in primary islet cells reduced GSIS, and the effect of BMP4 on GSIS was lost in islets from calbindin1 (Calb1) knockout mice. CONCLUSIONS/INTERPRETATION: We found BMP4 treatment to markedly inhibit GSIS from rodent pancreatic islets in a calbindin1-dependent manner. Calbindin1 is suggested to mediate the effect of BMP4 by buffering Ca(2+) and decreasing Ca(2+) channel activity, resulting in diminished insulin exocytosis. Both BMP4 and calbindin1 are potential pharmacological targets for the treatment of beta cell dysfunction.
Asunto(s)
Proteína Morfogenética Ósea 4/metabolismo , Calbindina 1/metabolismo , Calcio/metabolismo , Células Secretoras de Insulina/citología , Insulina/metabolismo , Animales , Calbindina 1/genética , Fenómenos Electrofisiológicos , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Secreción de Insulina , Islotes Pancreáticos/citología , Masculino , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Regulación hacia ArribaRESUMEN
AIMS/HYPOTHESIS: Impairment of beta cell mass and function is evident in both type 1 and type 2 diabetes. In healthy physiological conditions pancreatic beta cells adapt to the body's increasing insulin requirements by proliferation and improved function. We hypothesised that during the development of diabetes, there is an increase in the expression of inhibitory factors that prevent the beta cells from adapting to the increased need for insulin. We evaluated the effects of bone morphogenetic protein (BMP) 2 and -4 on beta cells. METHODS: The effects of BMP2 and -4 on beta cell proliferation, apoptosis, gene expression and insulin release were studied in isolated islets of Langerhans from rats, mice and humans. The expression of BMPs was analysed by immunocytochemistry and real-time PCR. The role of endogenous BMP was investigated using a soluble and neutralising form of the BMP receptor 1A. RESULTS: BMP2 and -4 were found to inhibit basal as well as growth factor-stimulated proliferation of primary beta cells from rats and mice. Bmp2 and Bmp4 mRNA and protein were expressed in islets and regulated by inflammatory cytokines. Neutralisation of endogenous BMP activity resulted in enhanced proliferation of rodent beta cells. The expression of Id mRNAs was induced by BMP4 in rat and human islets. Finally, glucose-induced insulin secretion was significantly impaired in rodent and human islets pre-treated with BMP4, and inhibition of BMP activity resulted in enhanced insulin release. CONCLUSIONS/INTERPRETATION: These data show that BMP2 and -4 exert inhibitory actions on beta cells in vitro and suggest that BMPs exert regulatory roles of beta cell growth and function.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteína Morfogenética Ósea 2/farmacología , Proteína Morfogenética Ósea 4/farmacología , Proliferación Celular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Células Secretoras de Insulina/efectos de los fármacos , Animales , Células Cultivadas , Insulina/metabolismo , Células Secretoras de Insulina/fisiología , Ratones , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
Seven-transmembrane receptors (7TMRs) signal through the well described heterotrimeric G proteins but can also activate G protein-independent signaling pathways of which the impact and complexity are less understood. The angiotensin II type 1 receptor (AT(1)R) is a prototypical 7TMR and an important drug target in cardiovascular diseases. "Biased agonists" with intrinsic "functional selectivity" that simultaneously blocks Galpha(q) protein activity and activates G protein-independent pathways of the AT(1)R confer important perspectives in treatment of cardiovascular diseases. In this study, we performed a global quantitative phosphoproteomics analysis of the AT(1)R signaling network. We analyzed ligand-stimulated SILAC (stable isotope labeling by amino acids in cell culture) cells by high resolution (LTQ-Orbitrap) MS and compared the phosphoproteomes of the AT(1)R agonist angiotensin II and the biased agonist [Sar(1),Ile(4),Ile(8)]angiotensin II (SII angiotensin II), which only activates the Galpha(q) protein-independent signaling. We quantified more than 10,000 phosphorylation sites of which 1183 were regulated by angiotensin II or its analogue SII angiotensin II. 36% of the AT(1)R-regulated phosphorylations were regulated by SII angiotensin II. Analysis of phosphorylation site patterns showed a striking distinction between protein kinases activated by Galpha(q) protein-dependent and -independent mechanisms, and we now place protein kinase D as a key protein involved in both Galpha(q)-dependent and -independent AT(1)R signaling. This study provides substantial novel insight into angiotensin II signal transduction and is the first study dissecting the differences between a full agonist and a biased agonist from a 7TMR on a systems-wide scale. Importantly, it reveals a previously unappreciated diversity and quantity of Galpha(q) protein-independent signaling and uncovers novel signaling pathways. We foresee that the amount and diversity of G protein-independent signaling may be more pronounced than previously recognized for other 7TMRs as well. Quantitative mass spectrometry is a promising tool for evaluation of the signaling properties of biased agonists to other receptors in the future.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Fosfoproteínas/análisis , Proteoma/análisis , Receptor de Angiotensina Tipo 1 , Secuencia de Aminoácidos , Línea Celular , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Humanos , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Receptor de Angiotensina Tipo 1/agonistas , Receptor de Angiotensina Tipo 1/metabolismo , Transducción de Señal/fisiologíaRESUMEN
The hypocretin/orexin system regulates arousal through central nervous system mechanisms and plays an important role in sleep, wakefulness and energy homeostasis. It is unclear whether hypocretin peptides are also present in blood due to difficulties in measuring reliable and reproducible levels of the peptides in blood samples. Lack of hypocretin signalling causes the sleep disorder narcolepsy type 1, and low concentration of cerebrospinal fluid hypocretin-1/orexin-A peptide is a hallmark of the disease. This measurement has high diagnostic value, but performing a lumbar puncture is not without discomfort and possible complications for the patient. A blood-based test to assess hypocretin-1 deficiency would therefore be of obvious benefit. We here demonstrate that heating plasma or serum samples to 65°C for 30 min at pH 8 significantly increases hypocretin-1 immunoreactivity enabling stable and reproducible measurement of hypocretin-1 in blood samples. Specificity of the signal was verified by high-performance liquid chromatography and by measuring blood samples from mice lacking hypocretin. Unspecific background signal in the assay was high. Using our method, we show that hypocretin-1 immunoreactivity in blood samples from narcolepsy type 1 patients does not differ from the levels detected in control samples. The data presented here suggest that hypocretin-1 is present in the blood stream in the low picograms per millilitres range and that peripheral hypocretin-1 concentrations are unchanged in narcolepsy type 1.
RESUMEN
Several cross-sectional studies have shown hair cortisol concentration to be associated with adiposity, but the relationship between hair cortisol concentration and longitudinal changes in measures of adiposity are largely unknown. We included 786 adults from the NoHoW trial, who had achieved a successful weight loss of ≥5% and had a body mass index of ≥25 kg/m2 prior to losing weight. Hair cortisol concentration (pg/mg hair) was measured at baseline and after 12 months. Body weight and body fat percentage were measured at baseline, 6-month, 12-month and 18-month visits. Participants weighed themselves at home ≥2 weekly using a Wi-Fi scale for the 18-month study duration, from which body weight variability was estimated using linear and non-linear approaches. Regression models were conducted to examine log hair cortisol concentration and change in log hair cortisol concentration as predictors of changes in body weight, change in body fat percentage and body weight variability. After adjustment for lifestyle and demographic factors, no associations between baseline log hair cortisol concentration and outcome measures were observed. Similar results were seen when analysing the association between 12-month concurrent development in log hair cortisol concentration and outcomes. However, an initial 12-month increase in log hair cortisol concentration was associated with a higher subsequent body weight variability between month 12 and 18, based on deviations from a nonlinear trend (ß: 0.02% per unit increase in log hair cortisol concentration [95% CI: 0.00, 0.04]; P=0.016). Our data suggest that an association between hair cortisol concentration and subsequent change in body weight or body fat percentage is absent or marginal, but that an increase in hair cortisol concentration during a 12-month weight loss maintenance effort may predict a slightly higher subsequent 6-months body weight variability. Clinical Trial Registration: ISRCTN registry, identifier ISRCTN88405328.
Asunto(s)
Biomarcadores/análisis , Índice de Masa Corporal , Peso Corporal , Cabello/metabolismo , Hidrocortisona/metabolismo , Estrés Psicológico/fisiopatología , Pérdida de Peso , Adulto , Estudios Transversales , Femenino , Estudios de Seguimiento , Cabello/química , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Pronóstico , Estudios ProspectivosRESUMEN
Exposure to ionizing radiation increases the risk of chronic metabolic disorders such as insulin resistance and type 2 diabetes later in life. We hypothesized that irradiation reprograms the epigenome of metabolic progenitor cells, which could account for impaired metabolism after cancer treatment. C57Bl/6 mice were treated with a single dose of irradiation and subjected to high-fat diet (HFD). RNA sequencing and reduced representation bisulfite sequencing were used to create transcriptomic and epigenomic profiles of preadipocytes and skeletal muscle satellite cells collected from irradiated mice. Mice subjected to total body irradiation showed alterations in glucose metabolism and, when challenged with HFD, marked hyperinsulinemia. Insulin signaling was chronically disrupted in skeletal muscle and adipose progenitor cells collected from irradiated mice and differentiated in culture. Epigenomic profiling of skeletal muscle and adipose progenitor cells from irradiated animals revealed substantial DNA methylation changes, notably for genes regulating the cell cycle, glucose/lipid metabolism, and expression of epigenetic modifiers. Our results show that total body irradiation alters intracellular signaling and epigenetic pathways regulating cell proliferation and differentiation of skeletal muscle and adipose progenitor cells and provide a possible mechanism by which irradiation used in cancer treatment increases the risk for metabolic disease later in life.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Músculo Esquelético/metabolismo , Radiación Ionizante , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Animales , Peso Corporal/efectos de los fármacos , Peso Corporal/efectos de la radiación , Células Cultivadas , Biología Computacional , Epigenómica , Immunoblotting , Resistencia a la Insulina/efectos de la radiación , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/efectos de los fármacos , Especies Reactivas de Oxígeno , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ARNRESUMEN
The development of resistance in tamoxifen-treated breast cancer patients and the estrogenic side effects of tamoxifen have lead to the design of many new drugs. The new SERM arzoxifene and its active metabolite desmethylarzoxifene (ARZm) inhibits growth of breast cancer cells and has less estrogenic effects than tamoxifen on gene expression. A cell line with acquired resistance to ARZm (MCF-7/ARZm(R)-1) was established from MCF-7 cells. MCF-7/ARZm(R)-1 cells responded to treatment with tamoxifen and the pure antiestrogen ICI 182,7870. The estrogen receptor alpha (ERalpha) level in MCF-7/ARZm(R)-1 cells was lower than in MCF-7 cells due to a destabilization of the receptor by ARZm. A significant reduction in the mRNA and protein level of some estrogen-regulated genes was observed in MCF-7/ARZm(R)-1 compared to MCF-7. However, both the level of the ERalpha and several ER-regulated gene products increased towards parental MCF-7 level upon withdrawal from ARZm, concomitant with an increase in the sensitivity of MCF-7/ARZm(R)-1 cells to ARZm treatment. These data show that ARZm resistant cells remain sensitive to treatment with both tamoxifen and to ICI 182,780. Furthermore, the partial reversion to ARZm sensitivity upon withdrawal of the SERM suggests that patients may benefit from a rechallenge with ARZm.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos , Estradiol/análogos & derivados , Piperidinas/farmacología , Tiofenos/farmacología , Neoplasias de la Mama/genética , Catepsina D/metabolismo , División Celular/efectos de los fármacos , Línea Celular Tumoral , Estradiol/farmacología , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Fulvestrant , Histona Acetiltransferasas , Humanos , Proteínas Nucleares/metabolismo , Co-Represor 1 de Receptor Nuclear , Coactivador 1 de Receptor Nuclear , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/metabolismo , Receptores de Progesterona/metabolismo , Proteínas Represoras/metabolismo , Tamoxifeno/farmacología , Factores de Transcripción/metabolismoRESUMEN
During the development of diabetes ß-cells are exposed to elevated concentrations of proinflammatory cytokines, TNFα and IL1ß, which in vitro induce ß-cell death. The class B G-protein-coupled receptors (GPCRs): corticotropin-releasing factor receptor 1 (CRFR1) and CRFR2 are expressed in pancreatic islets. As downstream signaling by other class B GPCRs can protect against cytokine-induced ß-cell apoptosis, we evaluated the protective potential of CRFR activation in ß-cells in a pro-inflammatory setting. CRFR1/CRFR2 ligands activated AKT and CRFR1 signaling and reduced apoptosis in human islets. In rat and mouse insulin-secreting cell lines (INS-1 and MIN6), CRFR1 agonists upregulated insulin receptor substrate 2 (IRS2) expression, increased AKT activation, counteracted the cytokine-mediated decrease in BAD phosphorylation, and inhibited apoptosis. The anti-apoptotic signaling was dependent on prolonged exposure to corticotropin-releasing factor family peptides and followed PKA-mediated IRS2 upregulation. This indicates that CRFR signaling counteracts proinflammatory cytokine-mediated apoptotic pathways through upregulation of survival signaling in ß-cells. Interestingly, CRFR signaling also counteracted basal apoptosis in both cultured INS-1 cells and intact human islets.
Asunto(s)
Apoptosis/efectos de los fármacos , Hormona Liberadora de Corticotropina/farmacología , Citocinas/efectos adversos , Citoprotección/efectos de los fármacos , Células Secretoras de Insulina/efectos de los fármacos , Receptores de Hormona Liberadora de Corticotropina/agonistas , Animales , Muerte Celular/efectos de los fármacos , Células Cultivadas , Humanos , Células Secretoras de Insulina/fisiología , Interleucina-1beta/efectos adversos , Ratones , Ratas , Factor de Necrosis Tumoral alfa/efectos adversosRESUMEN
Recent progress in the understanding of seven-transmembrane receptor (7TMR) signalling has promoted the development of a new generation of pathway selective ligands. The angiotensin II type I receptor (AT1aR) is one of the most studied 7TMRs with respect to selective activation of the ß-arrestin dependent signalling. Two complimentary global phosphoproteomics studies have analyzed the complex signalling induced by the AT1aR. Here we integrate the data sets from these studies and perform a joint analysis using a novel method for prediction of differential kinase activity from phosphoproteomics data. The method builds upon NetworKIN, which applies sophisticated linear motif analysis in combination with contextual network modelling to predict kinase-substrate associations with high accuracy and sensitivity. These predictions form the basis for subsequently nonparametric statistical analysis to identify likely activated kinases. This suggested that AT1aR-dependent signalling activates 48 of the 285 kinases detected in HEK293 cells. Of these, Aurora B, CLK3 and PKG1 have not previously been described in the pathway whereas others, such as PKA, PKB and PKC, are well known. In summary, we have developed a new method for kinase-centric analysis of phosphoproteomes to pinpoint differential kinase activity in large-scale data sets.
Asunto(s)
Receptores de Angiotensina/metabolismo , Transducción de Señal/fisiología , Bases de Datos de Proteínas , Células HEK293 , Humanos , Fosforilación , Estructura Terciaria de Proteína , Análisis de Secuencia de ProteínaRESUMEN
The angiotensin II type 1 receptor (AT(1)R) is known to signal through heterotrimeric G proteins, and Gαq protein-independent signalling has only recently gained appreciation for profound impact on a diverse range of biological functions. ß-Arrestins, among other central mediators of Gαq protein-independent signalling from the AT(1)R interact with transcriptional regulators and promote phosphorylation of nuclear proteins. However, the relative contribution of Gαq protein-independent signalling in AT(1)R mediated transcriptional regulation remains elusive. We here present a comprehensive comparative analysis of Gαq protein-dependent and -independent regulation of AT(1)R mediated gene expression. We found angiotensin II to regulate 212 genes, whereas Gαq-independent signalling obtained with the biased agonist, SII angiotensin II only regulated few genes. Interestingly, SII angiotensin II, like Ang II vastly potentiated ß2-adrenergic receptor-stimulated gene expression. These novel findings indicate that the Gαq protein-independent signalling mainly modifies the transcriptional response governed by other signalling pathways, while direct induction of gene expression by the AT(1)R is dependent on classical Gαq protein activation.