RESUMEN
Control of fatty acid storage and release in adipose tissue is fundamental in energy homeostasis and the development of obesity and type 2 diabetes. We here take the whole signalling network into account to identify how insulin and ß-adrenergic stimulation in concert controls lipolysis in mature subcutaneous adipocytes obtained from non-diabetic and, in parallel, type 2 diabetic women. We report that, and show how, the anti-lipolytic effect of insulin can be fully explained by protein kinase B (PKB/Akt)-dependent activation of the phosphodiesterase PDE3B. Through the same PKB-dependent pathway ß-adrenergic receptor signalling, via cAMP and PI3Kα, is anti-lipolytic and inhibits its own stimulation of lipolysis by 50%. Through this pathway both insulin and ß-adrenergic signalling control phosphorylation of FOXO1. The dose-response of lipolysis is bell-shaped, such that insulin is anti-lipolytic at low concentrations, but at higher concentrations of insulin lipolysis was increasingly restored due to inhibition of PDE3B. The control of lipolysis was not altered in adipocytes from diabetic individuals. However, the release of fatty acids was increased by 50% in diabetes due to reduced reesterification of lipolytically liberated fatty acids. In conclusion, our results reveal mechanisms of control by insulin and ß-adrenergic stimulation - in human adipocytes - that define a network of checks and balances ensuring robust control to secure uninterrupted supply of fatty acids without reaching concentrations that put cellular integrity at risk. Moreover, our results define how selective insulin resistance leave lipolytic control by insulin unaltered in diabetes, while the fatty acid release is substantially increased.
Asunto(s)
Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Insulina/farmacología , Lipólisis , Receptores Adrenérgicos beta/metabolismo , Adipocitos/citología , Tejido Adiposo/citología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Células Cultivadas , Femenino , Humanos , Lipólisis/efectos de los fármacos , Lipólisis/fisiología , Persona de Mediana Edad , Obesidad/metabolismo , Adulto JovenRESUMEN
Type 2 diabetes is characterized by insulin resistance in the expanding adipose tissue of obesity. The insulin resistance manifests in human adipocytes as system-wide impairment of insulin signalling. An exception is the regulation of transcription factor FOXO1 (forkhead box protein O1), which is phosphorylated downstream of mTORC2 (mammalian/mechanistic target of rapamycin in complex with raptor) and is therefore not exhibiting impaired response to insulin. However, the abundance, and activity, of FOXO1 is reduced by half in adipocytes from patients with diabetes. To elucidate the effect of reduced FOXO1 activity, we here transduced human adipocytes with a dominant-negative construct of FOXO1 (DN-FOXO1). Inhibition of FOXO1 reduced the abundance of insulin receptor, glucose transporter-4, ribosomal protein S6, mTOR and raptor. Functionally, inhibition of FOXO1 induced an insulin-resistant state network-wide, a state that qualitatively and quantitatively mimicked adipocytes from patients with type 2 diabetes. In contrast, and in accordance with these effects of DN-FOXO1, overexpression of wild-type FOXO1 appeared to augment insulin signalling. We combined experimental data with mathematical modelling to show that the impaired insulin signalling in FOXO1-inhibited cells to a large extent can be explained by reduced mTORC1 activity - a mechanism that defines much of the diabetic state in human adipocytes. Our findings demonstrate that FOXO1 is critical for maintaining normal insulin signalling of human adipocytes.
Asunto(s)
Adipocitos/patología , Diabetes Mellitus Tipo 2/fisiopatología , Proteína Forkhead Box O1/antagonistas & inhibidores , Regulación de la Expresión Génica , Resistencia a la Insulina , Insulina/metabolismo , Adipocitos/metabolismo , Adulto , Anciano , Antígenos CD/metabolismo , Células Cultivadas , Femenino , Humanos , Persona de Mediana Edad , Fosforilación , Receptor de Insulina/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Insulin resistance is a major aspect of type 2 diabetes (T2D), which results from impaired insulin signaling in target cells. Signaling to regulate forkhead box protein O1 (FOXO1) may be the most important mechanism for insulin to control transcription. Despite this, little is known about how insulin regulates FOXO1 and how FOXO1 may contribute to insulin resistance in adipocytes, which are the most critical cell type in the development of insulin resistance. We report a detailed mechanistic analysis of insulin control of FOXO1 in human adipocytes obtained from non-diabetic subjects and from patients with T2D. We show that FOXO1 is mainly phosphorylated through mTORC2-mediated phosphorylation of protein kinase B at Ser(473) and that this mechanism is unperturbed in T2D. We also demonstrate a cross-talk from the MAPK branch of insulin signaling to stimulate phosphorylation of FOXO1. The cellular abundance and consequently activity of FOXO1 are halved in T2D. Interestingly, inhibition of mTORC1 with rapamycin reduces the abundance of FOXO1 to the levels in T2D. This suggests that the reduction of the concentration of FOXO1 is a consequence of attenuation of mTORC1, which defines much of the diabetic state in human adipocytes. We integrate insulin control of FOXO1 in a network-wide mathematical model of insulin signaling dynamics based on compatible data from human adipocytes. The diabetic state is network-wide explained by attenuation of an mTORC1-to-insulin receptor substrate-1 (IRS1) feedback and reduced abundances of insulin receptor, GLUT4, AS160, ribosomal protein S6, and FOXO1. The model demonstrates that attenuation of the mTORC1-to-IRS1 feedback is a major mechanism of insulin resistance in the diabetic state.
Asunto(s)
Adipocitos/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Proteína Forkhead Box O1/metabolismo , Insulina/metabolismo , Modelos Biológicos , Transducción de Señal , Adipocitos/patología , Células Cultivadas , Diabetes Mellitus Tipo 2/patología , Femenino , Proteínas Activadoras de GTPasa/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Humanos , Proteínas Sustrato del Receptor de Insulina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos/metabolismo , Fosforilación , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The ubiquitously expressed IQ motif-containing GTPase activating protein-1 (IQGAP1) is a scaffolding protein implicated in an array of cellular functions, in particular by binding to cytoskeletal elements and signaling proteins. A role of IQGAP1 in adipocytes has not been reported. We therefore investigated the cellular IQGAP1 interactome in primary human adipocytes. Immunoprecipitation and quantitative mass spectrometry identified caveolae and caveolae-associated proteins as the major IQGAP1 interactors alongside cytoskeletal proteins. We confirmed co-localization of IQGAP1 with the defining caveolar marker protein caveolin-1 by confocal microscopy and proximity ligation assay. Most interestingly, insulin enhanced the number of IQGAP1 interactions with caveolin-1 by five-fold. Moreover, we found a significantly reduced abundance of IQGAP1 in adipocytes from patients with type 2 diabetes compared with cells from nondiabetic control subjects. Both the abundance of IQGAP1 protein and mRNA were reduced, indicating a transcriptional defect in diabetes. Our findings suggest a novel role of IQGAP1 in insulin-regulated interaction between caveolae and cytoskeletal elements of the adipocyte, and that this is quelled in the diabetic state.
Asunto(s)
Adipocitos/metabolismo , Caveolas/metabolismo , Citoesqueleto/metabolismo , Insulina/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Adipocitos/citología , Diabetes Mellitus Tipo 2/metabolismo , Humanos , FosforilaciónRESUMEN
The response to insulin is impaired in type 2 diabetes. Much information is available about insulin signaling, but understanding of the cellular mechanisms causing impaired signaling and insulin resistance is hampered by fragmented data, mainly obtained from different cell lines and animals. We have collected quantitative and systems-wide dynamic data on insulin signaling in primary adipocytes and compared cells isolated from healthy and diabetic individuals. Mathematical modeling and experimental verification identified mechanisms of insulin control of the MAPKs ERK1/2. We found that in human adipocytes, insulin stimulates phosphorylation of the ribosomal protein S6 and hence protein synthesis about equally via ERK1/2 and mTORC1. Using mathematical modeling, we examined the signaling network as a whole and show that a single mechanism can explain the insulin resistance of type 2 diabetes throughout the network, involving signaling both through IRS1, PKB, and mTOR and via ERK1/2 to the nuclear transcription factor Elk1. The most important part of the insulin resistance mechanism is an attenuated feedback from the protein kinase mTORC1 to IRS1, which spreads signal attenuation to all parts of the insulin signaling network. Experimental inhibition of mTORC1 using rapamycin in adipocytes from non-diabetic individuals induced and thus confirmed the predicted network-wide insulin resistance.
Asunto(s)
Adipocitos/metabolismo , Complicaciones de la Diabetes/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Resistencia a la Insulina , Sistema de Señalización de MAP Quinasas , Obesidad/metabolismo , Adipocitos/patología , Adulto , Anciano , Complicaciones de la Diabetes/genética , Complicaciones de la Diabetes/patología , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Femenino , Humanos , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Persona de Mediana Edad , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Modelos Biológicos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Obesidad/genética , Obesidad/patología , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Type 2 diabetes originates in an expanding adipose tissue that for unknown reasons becomes insulin resistant. Insulin resistance reflects impairments in insulin signaling, but mechanisms involved are unclear because current research is fragmented. We report a systems level mechanistic understanding of insulin resistance, using systems wide and internally consistent data from human adipocytes. Based on quantitative steady-state and dynamic time course data on signaling intermediaries, normally and in diabetes, we developed a dynamic mathematical model of insulin signaling. The model structure and parameters are identical in the normal and diabetic states of the model, except for three parameters that change in diabetes: (i) reduced concentration of insulin receptor, (ii) reduced concentration of insulin-regulated glucose transporter GLUT4, and (iii) changed feedback from mammalian target of rapamycin in complex with raptor (mTORC1). Modeling reveals that at the core of insulin resistance in human adipocytes is attenuation of a positive feedback from mTORC1 to the insulin receptor substrate-1, which explains reduced sensitivity and signal strength throughout the signaling network. Model simulations with inhibition of mTORC1 are comparable with experimental data on inhibition of mTORC1 using rapamycin in human adipocytes. We demonstrate the potential of the model for identification of drug targets, e.g. increasing the feedback restores insulin signaling, both at the cellular level and, using a multilevel model, at the whole body level. Our findings suggest that insulin resistance in an expanded adipose tissue results from cell growth restriction to prevent cell necrosis.
Asunto(s)
Adipocitos/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Resistencia a la Insulina , Insulina/metabolismo , Complejos Multiproteicos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Adipocitos/citología , Femenino , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Humanos , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Metformina/farmacología , Modelos Teóricos , Músculos/metabolismo , Necrosis , Obesidad/metabolismo , Sobrepeso , Receptor de Insulina/metabolismo , Transducción de Señal , Piel/metabolismoRESUMEN
Type 2 diabetes is a metabolic disease that profoundly affects energy homeostasis. The disease involves failure at several levels and subsystems and is characterized by insulin resistance in target cells and tissues (i.e. by impaired intracellular insulin signaling). We have previously used an iterative experimental-theoretical approach to unravel the early insulin signaling events in primary human adipocytes. That study, like most insulin signaling studies, is based on in vitro experimental examination of cells, and the in vivo relevance of such studies for human beings has not been systematically examined. Herein, we develop a hierarchical model of the adipose tissue, which links intracellular insulin control of glucose transport in human primary adipocytes with whole-body glucose homeostasis. An iterative approach between experiments and minimal modeling allowed us to conclude that it is not possible to scale up the experimentally determined glucose uptake by the isolated adipocytes to match the glucose uptake profile of the adipose tissue in vivo. However, a model that additionally includes insulin effects on blood flow in the adipose tissue and GLUT4 translocation due to cell handling can explain all data, but neither of these additions is sufficient independently. We also extend the minimal model to include hierarchical dynamic links to more detailed models (both to our own models and to those by others), which act as submodules that can be turned on or off. The resulting multilevel hierarchical model can merge detailed results on different subsystems into a coherent understanding of whole-body glucose homeostasis. This hierarchical modeling can potentially create bridges between other experimental model systems and the in vivo human situation and offers a framework for systematic evaluation of the physiological relevance of in vitro obtained molecular/cellular experimental data.
Asunto(s)
Glucosa/metabolismo , Homeostasis , Insulina/metabolismo , Modelos Biológicos , Transducción de Señal , Adipocitos/metabolismo , Adulto , Anciano , Envejecimiento/metabolismo , Transporte Biológico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Caracteres SexualesRESUMEN
Much data in the scientific literature demonstrate a fundamental involvement of caveolae in insulin action, although particular aspects remain matters of debate. The insulin receptor and part of the downstream signalling mediators are localized in or recruited to caveolae. Moreover, as part of the signalling, insulin receptors are rapidly endocytosed by caveolae in response to the hormone. The insulin regulated glucose transporter GLUT4 appears to localize to caveolae after insulin-stimulated translocation to the plasma membrane, while the endocytosis of GLUT4 may involve a clathrin-mediated process. Insulin resistance due to dysfunction of insulin signalling in target tissues is a primary cornerstone of Type 2 diabetes. Lack of caveolae makes animals and human beings insulin resistant, but there is presently no evidence that caveolae play a role in the pathogenesis of insulin resistance in obesity and Type 2 diabetes.
Asunto(s)
Caveolas/metabolismo , Caveolinas/metabolismo , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Insulina/metabolismo , Transducción de Señal , Animales , Glucosa/metabolismo , HumanosRESUMEN
OBJECTIVE: Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) transports Ca2+ from the cytosol into the endoplasmic retitculum (ER) and is essential for appropriate regulation of intracellular Ca2+ homeostasis. The objective of this study was to test the hypothesis that SERCA pumps are involved in the regulation of white adipocyte hormone secretion and other aspects of adipose tissue function and that this control is disturbed in obesity-induced type-2 diabetes. METHODS: SERCA expression was measured in isolated human and mouse adipocytes as well as in whole mouse adipose tissue by Western blot and RT-qPCR. To test the significance of SERCA2 in adipocyte functionality and whole-body metabolism, we generated adipocyte-specific SERCA2 knockout mice. The mice were metabolically phenotyped by glucose tolerance and tracer studies, histological analyses, measurements of glucose-stimulated insulin release in isolated islets, and gene/protein expression analyses. We also tested the effect of pharmacological SERCA inhibition and genetic SERCA2 ablation in cultured adipocytes. Intracellular and mitochondrial Ca2+ levels were recorded with dual-wavelength ratio imaging and mitochondrial function was assessed by Seahorse technology. RESULTS: We demonstrate that SERCA2 is downregulated in white adipocytes from patients with obesity and type-2 diabetes as well as in adipocytes from diet-induced obese mice. SERCA2-ablated adipocytes display disturbed Ca2+ homeostasis associated with upregulated ER stress markers and impaired hormone release. These adipocyte alterations are linked to mild lipodystrophy, reduced adiponectin levels, and impaired glucose tolerance. Interestingly, adipocyte-specific SERCA2 ablation leads to increased glucose uptake in white adipose tissue while the glucose uptake is reduced in brown adipose tissue. This dichotomous effect on glucose uptake is due to differently regulated mitochondrial function. In white adipocytes, SERCA2 deficiency triggers an adaptive increase in fibroblast growth factor 21 (FGF21), increased mitochondrial uncoupling protein 1 (UCP1) levels, and increased oxygen consumption rate (OCR). In contrast, brown SERCA2 null adipocytes display reduced OCR despite increased mitochondrial content and UCP1 levels compared to wild type controls. CONCLUSIONS: Our data suggest causal links between reduced white adipocyte SERCA2 levels, deranged adipocyte Ca2+ homeostasis, adipose tissue dysfunction and type-2 diabetes.
Asunto(s)
Tejido Adiposo Pardo , Diabetes Mellitus Tipo 2 , Adipocitos Marrones/metabolismo , Tejido Adiposo Pardo/metabolismo , Animales , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Hormonas/metabolismo , Humanos , Ratones , Obesidad/metabolismoRESUMEN
Insulin and other hormones control target cells through a network of signal-mediating molecules. Such networks are extremely complex due to multiple feedback loops in combination with redundancy, shared signal mediators, and cross-talk between signal pathways. We present a novel framework that integrates experimental work and mathematical modeling to quantitatively characterize the role and relation between co-existing submechanisms in complex signaling networks. The approach is independent of knowing or uniquely estimating model parameters because it only relies on (i) rejections and (ii) core predictions (uniquely identified properties in unidentifiable models). The power of our approach is demonstrated through numerous iterations between experiments, model-based data analyses, and theoretical predictions to characterize the relative role of co-existing feedbacks governing insulin signaling. We examined phosphorylation of the insulin receptor and insulin receptor substrate-1 and endocytosis of the receptor in response to various different experimental perturbations in primary human adipocytes. The analysis revealed that receptor endocytosis is necessary for two identified feedback mechanisms involving mass and information transfer, respectively. Experimental findings indicate that interfering with the feedback may substantially increase overall signaling strength, suggesting novel therapeutic targets for insulin resistance and type 2 diabetes. Because the central observations are present in other signaling networks, our results may indicate a general mechanism in hormonal control.
Asunto(s)
Retroalimentación Fisiológica/fisiología , Modelos Biológicos , Receptor de Insulina/metabolismo , Transducción de Señal/fisiología , Adipocitos/citología , Adipocitos/metabolismo , Células Cultivadas , Simulación por Computador , Endocitosis/fisiología , Humanos , Immunoblotting , Proteínas Sustrato del Receptor de Insulina/metabolismo , FosforilaciónRESUMEN
Lipolysis and the release of fatty acids to supply energy fuel to other organs, such as between meals, during exercise, and starvation, are fundamental functions of the adipose tissue. The intracellular lipolytic pathway in adipocytes is activated by adrenaline and noradrenaline, and inhibited by insulin. Circulating fatty acids are elevated in type 2 diabetic individuals. The mechanisms behind this elevation are not fully known, and to increase the knowledge a link between the systemic circulation and intracellular lipolysis is key. However, data on lipolysis and knowledge from in vitro systems have not been linked to corresponding in vivo data and knowledge in vivo. Here, we use mathematical modelling to provide such a link. We examine mechanisms of insulin action by combining in vivo and in vitro data into an integrated mathematical model that can explain all data. Furthermore, the model can describe independent data not used for training the model. We show the usefulness of the model by simulating new and more challenging experimental setups in silico, e.g. the extracellular concentration of fatty acids during an insulin clamp, and the difference in such simulations between individuals with and without type 2 diabetes. Our work provides a new platform for model-based analysis of adipose tissue lipolysis, under both non-diabetic and type 2 diabetic conditions.
Asunto(s)
Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Ácidos Grasos/metabolismo , Lipólisis/fisiología , Biología de Sistemas , Simulación por Computador , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Diabetes Mellitus Tipo 2/sangre , Ácidos Grasos/sangre , Humanos , Técnicas In Vitro , Insulina/metabolismo , Resistencia a la Insulina , Modelos Estadísticos , Modelos Teóricos , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Receptores Adrenérgicos beta/metabolismo , Transducción de Señal , Programas Informáticos , Triglicéridos/metabolismo , IncertidumbreRESUMEN
Type 2 diabetes (T2D) is strongly linked to obesity and an adipose tissue unresponsive to insulin. The insulin resistance is due to defective insulin signaling, but details remain largely unknown. We examined insulin signaling in adipocytes from T2D patients, and contrary to findings in animal studies, we observed attenuation of insulin activation of mammalian target of rapamycin (mTOR) in complex with raptor (mTORC1). As a consequence, mTORC1 downstream effects were also affected in T2D: feedback signaling by insulin to signal-mediator insulin receptor substrate-1 (IRS1) was attenuated, mitochondria were impaired and autophagy was strongly upregulated. There was concomitant autophagic destruction of mitochondria and lipofuscin particles, and a dependence on autophagy for ATP production. Conversely, mitochondrial dysfunction attenuated insulin activation of mTORC1, enhanced autophagy and attenuated feedback to IRS1. The overactive autophagy was associated with large numbers of cytosolic lipid droplets, a subset with colocalization of perlipin and the autophagy protein LC3/atg8, which can contribute to excessive fatty acid release. Patients with diagnoses of T2D and overweight were consecutively recruited from elective surgery, whereas controls did not have T2D. Results were validated in a cohort of patients without diabetes who exhibited a wide range of insulin sensitivities. Because mitochondrial dysfunction, inflammation, endoplasmic-reticulum stress and hypoxia all inactivate mTORC1, our results may suggest a unifying mechanism for the pathogenesis of insulin resistance in T2D, although the underlying causes might differ.
Asunto(s)
Adipocitos/metabolismo , Autofagia , Diabetes Mellitus Tipo 2/metabolismo , Obesidad/metabolismo , Factores de Transcripción/metabolismo , Adenosina Trifosfato/metabolismo , Adipocitos/patología , Adulto , Anciano , Citosol/metabolismo , Diabetes Mellitus Tipo 2/patología , Glucosa/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Insulina/metabolismo , Modelos Lineales , Metabolismo de los Lípidos , Lipofuscina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Microscopía Confocal , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Complejos Multiproteicos , Obesidad/patología , Fosforilación Oxidativa , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Proteínas , Transducción de Señal , Serina-Treonina Quinasas TOR , Factores de Transcripción/genéticaRESUMEN
In adipocytes, PDE3B (phosphodiesterase 3B) is an important regulatory effector in signalling pathways controlled by insulin and cAMP-increasing hormones. Stimulation of 3T3-L1 adipocytes with insulin or the beta3-adrenergic receptor agonist CL316243 (termed CL) indicated that insulin preferentially phosphorylated/activated PDE3B associated with internal membranes (endoplasmic reticulum/Golgi), whereas CL preferentially phosphorylated/activated PDE3B associated with caveolae. siRNA (small interfering RNA)-mediated KD (knockdown) of CAV-1 (caveolin-1) in 3T3-L1 adipocytes resulted in down-regulation of expression of membrane-associated PDE3B. Insulin-induced activation of PDE3B was reduced, whereas CL-mediated activation was almost totally abolished. Similar results were obtained in adipocytes from Cav-1-deficient mice. siRNA-mediated KD of CAV-1 in 3T3-L1 adipocytes also resulted in inhibition of CL-stimulated phosphorylation of HSL (hormone-sensitive lipase) and perilipin A, and of lipolysis. Superose 6 gel-filtration chromatography of solubilized membrane proteins from adipocytes stimulated with insulin or CL demonstrated the reversible assembly of distinct macromolecular complexes that contained 32P-phosphorylated PDE3B and signalling molecules thought to be involved in its activation. Insulin- and CL-induced macromolecular complexes were enriched in cholesterol, and contained certain common signalling proteins [14-3-3, PP2A (protein phosphatase 2A) and cav-1]. The complexes present in insulin-stimulated cells contained tyrosine-phosphorylated IRS-1 (insulin receptor substrate 1) and its downstream signalling proteins, whereas CL-activated complexes contained beta3-adrenergic receptor, PKA-RII [PKA (cAMP-dependent protein kinase)-regulatory subunit] and HSL. Insulin- and CL-mediated macromolecular complex formation was significantly inhibited by CAV-1 KD. These results suggest that cav-1 acts as a molecular chaperone or scaffolding molecule in cholesterol-rich lipid rafts that may be necessary for the proper stabilization and activation of PDE3B in response to CL and insulin.
Asunto(s)
Adipocitos/efectos de los fármacos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Dioxoles/farmacología , Insulina/farmacología , Células 3T3-L1 , Adipocitos/citología , Adipocitos/metabolismo , Agonistas Adrenérgicos beta/metabolismo , Agonistas Adrenérgicos beta/farmacología , Animales , Western Blotting , Caveolas/efectos de los fármacos , Caveolas/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Retículo Endoplásmico/enzimología , Activación Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica , Aparato de Golgi/enzimología , Lipólisis/efectos de los fármacos , Sustancias Macromoleculares/metabolismo , Ratones , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Especificidad por SustratoRESUMEN
Here we have investigated the role of the protein caveolin 1 (Cav1) and caveolae in the secretion of the white adipocyte hormone adiponectin. Using mouse primary subcutaneous adipocytes genetically depleted of Cav1, we show that the adiponectin secretion, stimulated either adrenergically or by insulin, is abrogated while basal (unstimulated) release of adiponectin is elevated. Adiponectin secretion is similarly affected in wildtype mouse and human adipocytes where the caveolae structure was chemically disrupted. The altered ex vivo secretion in adipocytes isolated from Cav1 null mice is accompanied by lowered serum levels of the high-molecular weight (HMW) form of adiponectin, whereas the total concentration of adiponectin is unaltered. Interestingly, levels of HMW adiponectin are maintained in adipose tissue from Cav1-depleted mice, signifying that a secretory defect is present. The gene expression of key regulatory proteins known to be involved in cAMP/adrenergically triggered adiponectin exocytosis (the beta-3-adrenergic receptor and exchange protein directly activated by cAMP) remains intact in Cav1 null adipocytes. Microscopy and fractionation studies indicate that adiponectin vesicles do not co-localise with Cav1 but that some vesicles are associated with a specific fraction of caveolae. Our studies propose that Cav1 has an important role in secretion of HMW adiponectin, even though adiponectin-containing vesicles are not obviously associated with this protein. We suggest that Cav1, and/or the caveolae domain, is essential for the organisation of signalling pathways involved in the regulation of HMW adiponectin exocytosis, a function that is disrupted in Cav1/caveolae-depleted adipocytes.
Asunto(s)
Adipocitos Blancos/metabolismo , Adiponectina/metabolismo , Caveolina 1/fisiología , Adiponectina/sangre , Adiponectina/genética , Adulto , Anciano , Animales , Caveolina 1/deficiencia , Membrana Celular/química , Dieta , Exocitosis/fisiología , Femenino , Expresión Génica/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Obesidad/etiología , Obesidad/metabolismoRESUMEN
Insulin resistance and type 2 diabetes (T2D) are closely linked to obesity. Numerous prospective studies have reported on weight gain, insulin resistance, and insulin signaling in experimental animals, but not in humans. We examined insulin signaling in adipocytes from lean volunteers, before and at the end of a 4-wk period of consuming a fast-food, high-calorie diet that led to weight gain. We also examined adipocytes from patients with T2D. During the high-calorie diet, subjects gained 10% body weight and 19% total body fat, but stayed lean (body mass index = 24.3 kg/m(2)) and developed moderate systemic insulin resistance. Similarly to the situation in T2D subjects, in subjects on the high-calorie diet, the amount of insulin receptors was reduced and phosphorylation of IRS1 at tyrosine and at serine-307 (human sequence, corresponding to murine serine-302) were impaired. The amount of insulin receptor substrate protein-1 (IRS1) and the phosphorylation of IRS1 at serine-312 (human sequence, corresponding to murine serine-307) were unaffected by the diet. Unlike the T2D subjects, in subjects on the high-calorie diet, likely owing to the ongoing weight-gain, phosphorylation of MAP-kinases ERK1/2 became hyperresponsive to insulin. To our knowledge this study is the first to investigate insulin signaling during overeating in humans, and it demonstrates that T2D effects on intracellular insulin signaling already occur after 4 wks of a high-calorie diet and that the effects in humans differ from those in laboratory animals.
Asunto(s)
Adipocitos/metabolismo , Hiperfagia/metabolismo , Resistencia a la Insulina/fisiología , Insulina/metabolismo , Delgadez/metabolismo , Absorciometría de Fotón , Adulto , Índice de Masa Corporal , Diabetes Mellitus Tipo 2/metabolismo , Dieta , Femenino , Humanos , Proteínas Sustrato del Receptor de Insulina/metabolismo , Masculino , Persona de Mediana Edad , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina/metabolismo , Transducción de Señal , Tirosina/metabolismoRESUMEN
Type 2 diabetes is characterized by insulin resistance of target organs, which is due to impaired insulin signal transduction. The skeleton of signaling mediators that provide for normal insulin action has been established. However, the detailed kinetics, and their mechanistic generation, remain incompletely understood. We measured time-courses in primary human adipocytes for the short-term phosphorylation dynamics of the insulin receptor (IR) and the IR substrate-1 in response to a step increase in insulin concentration. Both proteins exhibited a rapid transient overshoot in tyrosine phosphorylation, reaching maximum within 1 min, followed by an intermediate steady-state level after approximately 10 min. We used model-based hypothesis testing to evaluate three mechanistic explanations for this behavior: (A) phosphorylation and dephosphorylation of IR at the plasma membrane only; (B) the additional possibility for IR endocytosis; (C) the alternative additional possibility of feedback signals to IR from downstream intermediates. We concluded that (A) is not a satisfactory explanation; that (B) may serve as an explanation only if both internalization, dephosphorylation, and subsequent recycling are permitted; and that (C) is acceptable. These mechanistic insights cannot be obtained by mere inspection of the datasets, and they are rejections and thus stronger and more final conclusions than ordinary model predictions.
Asunto(s)
Adipocitos/metabolismo , Metabolismo Energético/fisiología , Insulina/metabolismo , Modelos Biológicos , Transducción de Señal/fisiología , Animales , Membrana Celular/metabolismo , Citosol/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Endocitosis , Retroalimentación Fisiológica/fisiología , Femenino , Homeostasis/fisiología , Humanos , Resistencia a la Insulina/fisiología , Ratones , Conejos , Receptor de Insulina/metabolismoRESUMEN
The mitochondrial enzyme 1-acyl-sn-glycerol-3-phosphate acyltransferase (mtGPAT1) catalyzes a rate-limiting step in triacylglycerol and glycerophospholipid biosynthesis, which can be modulated by protein kinases in cell free analyses. We report that treatment of primary rat adipocytes with insulin acutely affects the activity of mtGPAT1 by increasing V(MAX) and K(M) for the substrates glycerol-3-phosphate and palmitoyl-CoA. Proteolytic cleavage of isolated mitochondrial membranes and mass spectrometric peptide sequencing identify in vivo phosphorylation of serine 632 and serine 639 in mtGPAT1. These phosphorylation sites correspond to casein kinase-2 consensus sequences and are highly conserved in chordate animal, but not fly, fungal or plant, mtGPAT1.
Asunto(s)
Adipocitos/efectos de los fármacos , Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Insulina/farmacología , Mitocondrias/efectos de los fármacos , Proteínas Quinasas/metabolismo , Adipocitos/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Quinasa de la Caseína II/metabolismo , Catálisis , Glicerofosfolípidos/biosíntesis , Cinética , Espectrometría de Masas , Mitocondrias/enzimología , Datos de Secuencia Molecular , Fosforilación , Ratas , Ratas Sprague-Dawley , Serina/química , Serina/metabolismo , Triglicéridos/biosíntesisRESUMEN
Reduced sensitivity to insulin in adipose, muscle, and liver tissues is a hallmark of type 2 diabetes. Animal models and patients with type 2 diabetes exhibit elevated levels of circulating retinol-binding protein (RBP4), and RBP4 can induce insulin resistance in mice. However, little is known about how RBP4 affects insulin signaling. We examined the mechanisms of action of RBP4 in primary human adipocytes. RBP4-treated adipocytes exhibited the same molecular defects in insulin signaling, via IRS1 to MAP kinase, as in adipocytes from patients with type 2 diabetes. Without affecting autophosphorylation of the insulin receptor, RBP4 blocked the insulin-stimulated phosphorylation of IRS1 at serine (307) [corresponding to serine (302) in the murine sequence] and concomitantly increased the EC50 (from 0.5 to 2 nM) for insulin stimulation of IRS1 phosphorylation at tyrosine. The phosphorylation of IRS1 at serine (312) [corresponding to serine (307) in the murine sequence] was not affected in cells from diabetic patients and was also not affected by RBP4. The EC50 for insulin stimulation of downstream phosphorylation of MAP kinase ERK1/2 was increased (from 0.2 to 0.8 nM) by RBP4. We show that ERK1/2 phosphorylation is similarly impaired in adipocytes from patients with type 2 diabetes. However, the sensitivity to insulin for downstream signaling to control of protein kinase B and glucose uptake was not affected by RBP4. When insulin-resistant adipocytes from patients with type 2 diabetes were incubated with antibodies against RBP4, insulin-induced phosphorylation of IRS1 at serine (307) was normalized and the EC50 for insulin stimulation of ERK1/2 phosphorylation was reduced. Endogenous levels of RBP4 were markedly reduced in adipocytes from obese or type 2 diabetic subjects, whereas expression levels of RBP4 mRNA were unaffected. These findings indicate that RBP4 may be released from diabetic adipocytes and act locally to inhibit phosphorylation of IRS1 at serine (307), a phosphorylation site that may integrate nutrient sensing with insulin signaling.
Asunto(s)
Adipocitos/metabolismo , Insulina/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Plasmáticas de Unión al Retinol/fisiología , Adipocitos/enzimología , Humanos , Proteínas Sustrato del Receptor de Insulina , Fosforilación , Transducción de SeñalRESUMEN
The plasma membrane of cells functions as a barrier to the environment. Caveolae are minute invaginations of the membrane that selectively carry out the exchange of information and materials with the environment, by functioning as organizers of signal transduction and through endocytosis. Recent findings of uptake of different metabolites and of lipid metabolism occurring in caveolae, point to a new general function of caveolae. As gateways for the uptake of nutrients across the plasma membrane, and as platforms for the metabolic conversion of nutrients, especially in adipocytes, caveolae are now emerging as active centers for many aspects of intermediary metabolism, with implications for our understanding of obesity, diabetes and other metabolic disorders.
Asunto(s)
Adipocitos/metabolismo , Caveolas/metabolismo , Metabolismo Energético/fisiología , Metabolismo de los Lípidos/fisiología , Adipocitos/ultraestructura , Animales , Glucemia/metabolismo , Caveolas/ultraestructura , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Colesterol/metabolismo , Ácidos Grasos/metabolismo , Humanos , Microscopía Electrónica de Rastreo , Triglicéridos/metabolismoRESUMEN
Caveolae are noncoated invaginations of the plasma membrane that form in the presence of the protein caveolin. Caveolae are found in most cells, but are especially abundant in adipocytes. By high-resolution electron microscopy of plasma membrane sheets the detailed structure of individual caveolae of primary rat adipocytes was examined. Caveolin-1 and -2 binding was restricted to the membrane proximal region, such as the ducts or necks attaching the caveolar bulb to the membrane. This was confirmed by transfection with myc-tagged caveolin-1 and -2. Essentially the same results were obtained with human fibroblasts. Hence caveolin does not form the caveolar bulb in these cells, but rather the neck and may thus act to retain the caveolar constituents, indicating how caveolin participates in the formation of caveolae. Caveolae, randomly distributed over the plasma membrane, were very heterogeneous, varying in size between 25 and 150 nm. There was about one million caveolae in an adipocyte, which increased the surface area of the plasma membrane by 50%. Half of the caveolae, those larger than 50 nm, had access to the outside of the cell via ducts and 20-nm orifices at the cell surface. The rest of the caveolae, those smaller than 50 nm, were not open to the cell exterior. Cholesterol depletion destroyed both caveolae and the cell surface orifices.