RESUMEN
The two insulin receptor (IR) isoforms IR-A and IR-B are responsible for the pleiotropic actions of insulin and insulin-like growth factors. Consequently, changes in IR isoform expression and in the bioavailability of their ligands will impact on IR-mediated functions. Although alteration of IR isoform expression has been linked to insulin resistance, knowledge of IR isoform expression and mechanisms underlying tissue/cell-type-specific changes in metabolic disease are lacking. Using mouse models of obesity/diabetes and measuring the mRNA of the IR isoforms and mRNA/protein levels of total IR, we provide a data set of IR isoform expression pattern that documents changes in a tissue-dependent manner. Combining tissue fractionation and a new in situ mRNA hybridization technology to visualize the IR isoforms at cellular resolution, we explored the mechanism underlying the change in IR isoform expression in perigonadal adipose tissue, which is mainly caused by tissue remodelling, rather than by a shift in IR alternative splicing in a particular cell type, e.g. adipocytes.
Asunto(s)
Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 2/patología , Regulación de la Expresión Génica , Resistencia a la Insulina , Obesidad/complicaciones , Receptor de Insulina/metabolismo , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Empalme Alternativo , Animales , Diabetes Mellitus Experimental/etiología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/metabolismo , Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Isoformas de Proteínas , Receptor de Insulina/genética , Transducción de SeñalRESUMEN
Loss of pancreatic ß-cell function is a critical event in the pathophysiology of type 2 diabetes. However, studies of its underlying mechanisms as well as the discovery of novel targets and therapies have been hindered due to limitations in available experimental models. In this study we exploited the stable viability and function of standardized human islet microtissues to develop a disease-relevant, scalable, and reproducible model of ß-cell dysfunction by exposing them to long-term glucotoxicity and glucolipotoxicity. Moreover, by establishing a method for highly-efficient and homogeneous viral transduction, we were able to monitor the loss of functional ß-cell mass in vivo by transplanting reporter human islet microtissues into the anterior chamber of the eye of immune-deficient mice exposed to a diabetogenic diet for 12 weeks. This newly developed in vitro model as well as the described in vivo methodology represent a new set of tools that will facilitate the study of ß-cell failure in type 2 diabetes and would accelerate the discovery of novel therapeutic agents.
Asunto(s)
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Trasplante de Islotes Pancreáticos , Animales , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Xenoinjertos , Humanos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Masculino , Ratones Endogámicos NOD , Ratones NoqueadosRESUMEN
Although convincing in genetic models, the relevance of ß-cell insulin resistance in diet-induced type 2 diabetes (T2DM) remains unclear. Exemplified by diabetes-prone, male, C57B1/6J mice being fed different combinations of Western-style diet, we show that ß-cell insulin resistance occurs early during T2DM progression and is due to a combination of lipotoxicity and increased ß-cell workload. Within 8 wk of being fed a high-fat, high-sucrose diet, mice became obese, developed impaired insulin and glucose tolerances, and displayed noncompensatory insulin release, due, at least in part, to reduced expression of syntaxin-1A. Through reporter islets transplanted to the anterior chamber of the eye, we demonstrated a concomitant loss of functional ß-cell mass. When mice were changed from diabetogenic diet to normal chow diet, the diabetes phenotype was reversed, suggesting a remarkable plasticity of functional ß-cell mass in the early phase of T2DM development. Our data reinforce the relevance of diet composition as an environmental factor determining different routes of diabetes progression in a given genetic background. Employing the in vivo reporter islet-monitoring approach will allow researchers to define key times in the dynamics of reversible loss of functional ß-cell mass and, thus, to investigate the underlying, molecular mechanisms involved in the progression toward T2DM manifestation.-Paschen, M., Moede, T., Valladolid-Acebes, I., Leibiger, B., Moruzzi, N., Jacob, S., García-Prieto, C. F., Brismar, K., Leibiger, I. B., Berggren, P.-O. Diet-induced ß-cell insulin resistance results in reversible loss of functional ß-cell mass.
Asunto(s)
Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 2/patología , Dieta Alta en Grasa/efectos adversos , Sacarosa en la Dieta/efectos adversos , Resistencia a la Insulina , Células Secretoras de Insulina/patología , Insulina/metabolismo , Animales , Células Cultivadas , Diabetes Mellitus Experimental/etiología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Insulin resistance and ß-cell failure are the major defects in type 2 diabetes mellitus. However, the molecular mechanisms linking these two defects remain unknown. Elevated levels of apolipoprotein CIII (apoCIII) are associated not only with insulin resistance but also with cardiovascular disorders and inflammation. We now demonstrate that local apoCIII production is connected to pancreatic islet insulin resistance and ß-cell failure. An increase in islet apoCIII causes promotion of a local inflammatory milieu, increased mitochondrial metabolism, deranged regulation of ß-cell cytoplasmic free Ca(2+) concentration ([Ca(2+)]i) and apoptosis. Decreasing apoCIII in vivo results in improved glucose tolerance, and pancreatic apoCIII knockout islets transplanted into diabetic mice, with high systemic levels of the apolipoprotein, demonstrate a normal [Ca(2+)]i response pattern and no hallmarks of inflammation. Hence, under conditions of islet insulin resistance, locally produced apoCIII is an important diabetogenic factor involved in impairment of ß-cell function and may thus constitute a novel target for the treatment of type 2 diabetes mellitus.
Asunto(s)
Apolipoproteína C-III/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Resistencia a la Insulina/fisiología , Células Secretoras de Insulina/patología , Análisis de Varianza , Animales , Apolipoproteína C-III/genética , Western Blotting , Calcio/metabolismo , Línea Celular Tumoral , Inmunohistoquímica , Ratones , Ratones Noqueados , Microscopía Confocal , Mitocondrias/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The pancreatic islets of Langerhans consist of endocrine cells that secrete peptide hormones into the blood circulation in response to metabolic stimuli. When transplanted into the anterior chamber of the eye (ACE), pancreatic islets engraft and maintain morphological features of native islets as well as islet-specific vascularization and innervation patterns. In sufficient amounts, intraocular islets are able to maintain glucose homeostasis in diabetic mice. Islet organoids (pseudo-islets), which are formed by self-reassembly of islet cells following disaggregation and genetic manipulation, behave similarly to native islets. Here, we tested the hypothesis that genetically engineered intraocular islet organoids can serve as production sites for leptin. To test this hypothesis, we chose the leptin-deficient ob/ob mouse as a model system, which becomes severely obese, hyperinsulinemic, hyperglycemic, and insulin resistant. We generated a Tet-OFF-based beta-cell-specific adenoviral expression construct for mouse leptin, which allowed efficient transduction of native beta-cells, optical monitoring of leptin expression by co-expressed fluorescent proteins, and the possibility to switch-off leptin expression by treatment with doxycycline. Intraocular transplantation of islet organoids formed from transduced islet cells, which lack functional leptin receptors, to ob/ob mice allowed optical monitoring of leptin expression and ameliorated their metabolic phenotype by improving bodyweight, glucose tolerance, serum insulin, and C-peptide levels.
RESUMEN
Insulin resistance contributes to the development of cardio-vascular disease and diabetes. An important but unresolved task is to study the dynamics of insulin resistance in selective cell types of insulin target tissues in vivo. Here we present a novel technique to monitor insulin resistance dynamics non-invasively and longitudinally in vivo in a cell type-specific manner, exemplified by the pancreatic ß-cell situated within the micro-organ the islet of Langerhans. We utilize the anterior chamber of the eye (ACE) as a transplantation site and the cornea as a natural body-window to study the development and reversibility of insulin resistance. Engrafted islets in the ACE that express a FoxO1-GFP-based biosensor in their ß-cells, report on insulin resistance measured by fluorescence microscopy at single-cell resolution in the living mouse. This technique allows monitoring of cell type specific insulin sensitivity/resistance in real-time in the context of whole body insulin resistance during progression and intervention of disease.
Asunto(s)
Linaje de la Célula/genética , Rastreo Celular/métodos , Resistencia a la Insulina/genética , Células Secretoras de Insulina/metabolismo , Animales , Córnea/metabolismo , Córnea/patología , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/patología , Células Secretoras de Insulina/trasplante , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Ratones , Microscopía Fluorescente , Análisis de la Célula IndividualRESUMEN
Insulin resistance is a syndrome that affects multiple insulin target tissues, each having different biological functions regulated by insulin. A remaining question is to mechanistically explain how an insulin target cell/tissue can be insulin resistant in one biological function and insulin sensitive in another at the same time. Here, we provide evidence that in pancreatic ß cells, knockdown of PI3K-C2α expression results in rerouting of the insulin signal from insulin receptor (IR)-B/PI3K-C2α/PKB-mediated metabolic signaling to IR-B/Shc/ERK-mediated mitogenic signaling, which allows the ß cell to switch from a highly glucose-responsive, differentiated state to a proliferative state. Our data suggest the existence of IR-cascade-selective insulin resistance, which allows rerouting of the insulin signal within the same target cell. Hence, factors involved in the rerouting of the insulin signal represent tentative therapeutic targets in the treatment of insulin resistance.