RESUMEN
Islet vascularization is essential for intact islet function and glucose homeostasis. We have previously shown that primary cilia directly regulate insulin secretion. However, it remains unclear whether they are also implicated in islet vascularization. At eight weeks, murine Bbs4-/-islets show significantly lower intra-islet capillary density with enlarged diameters. Transplanted Bbs4-/- islets exhibit delayed re-vascularization and reduced vascular fenestration after engraftment, partially impairing vascular permeability and glucose delivery to ß-cells. We identified primary cilia on endothelial cells as the underlying cause of this regulation, via the vascular endothelial growth factor-A (VEGF-A)/VEGF receptor 2 (VEGFR2) pathway. In vitro silencing of ciliary genes in endothelial cells disrupts VEGF-A/VEGFR2 internalization and downstream signaling. Consequently, key features of angiogenesis including proliferation and migration are attenuated in human BBS4 silenced endothelial cells. We conclude that endothelial cell primary cilia regulate islet vascularization and vascular barrier function via the VEGF-A/VEGFR2 signaling pathway.
Asunto(s)
Células Endoteliales/fisiología , Islotes Pancreáticos/irrigación sanguínea , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular , Animales , Línea Celular , Femenino , Glucosa/metabolismo , Células HEK293 , Humanos , Islotes Pancreáticos/crecimiento & desarrollo , Ratones , Ratones Endogámicos , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Diabetes mellitus affects one in eleven adults worldwide. Most suffer from Type 2 Diabetes which features elevated blood glucose levels and an inability to adequately secrete or respond to insulin. Insulin producing ß-cells have primary cilia which are implicated in the regulation of glucose metabolism, insulin signaling and secretion. To better understand how ß-cell cilia affect glucose handling, we ablate cilia from mature ß-cells by deleting key cilia component Ift88. Here we report that glucose homeostasis and insulin secretion deteriorate over 12 weeks post-induction. Cilia/basal body components are required to suppress spontaneous auto-activation of EphA3 and hyper-phosphorylation of EphA receptors inhibits insulin secretion. In ß-cells, loss of cilia/basal body function leads to polarity defects and epithelial-to-mesenchymal transition. Defective insulin secretion from IFT88-depleted human islets and elevated pEPHA3 in islets from diabetic donors both point to a role for cilia/basal body proteins in human glucose homeostasis.
Asunto(s)
Cilios/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Endosomas/metabolismo , Glucosa/metabolismo , Homeostasis , Células Secretoras de Insulina/metabolismo , Receptores de la Familia Eph/metabolismo , Anciano , Animales , Glucemia , Prueba de Tolerancia a la Glucosa , Factores de Intercambio de Guanina Nucleótido , Humanos , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Neuropéptidos/metabolismo , Fosforilación , Receptor EphA3/genética , Receptor EphA3/metabolismo , Transducción de Señal , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Progressive decline of pancreatic beta cell function is central to the pathogenesis of type 2 diabetes. Protein phosphorylation regulates glucose-stimulated insulin secretion from beta cells, but how signaling networks are remodeled in diabetic islets in vivo remains unknown. Using high-sensitivity mass spectrometry-based proteomics, we quantified 6,500 proteins and 13,000 phosphopeptides in islets of obese diabetic mice and matched controls, revealing drastic remodeling of key kinase hubs and signaling pathways. Integration with a literature-derived signaling network implicated GSK3 kinase in the control of the beta cell-specific transcription factor PDX1. Deep phosphoproteomic analysis of human islets chronically treated with high glucose demonstrated a conserved glucotoxicity-dependent role of GSK3 kinase in regulating insulin secretion. Remarkably, the ability of beta cells to secrete insulin in response to glucose was rescued almost completely by pharmacological inhibition of GSK3. Thus, our resource enables investigation of mechanisms and drug targets in type 2 diabetes.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Proteínas de Homeodominio/metabolismo , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Transactivadores/metabolismo , Animales , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Glucógeno Sintasa Quinasa 3/genética , Proteínas de Homeodominio/genética , Humanos , Secreción de Insulina/genética , Células Secretoras de Insulina/química , Células Secretoras de Insulina/patología , Islotes Pancreáticos/química , Islotes Pancreáticos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfoproteínas/análisis , Fosfoproteínas/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Proteómica/métodos , Receptores de Leptina/genética , Transducción de Señal , Transactivadores/genéticaRESUMEN
An insufficient adaptive beta-cell compensation is a hallmark of type 2 diabetes (T2D). Primary cilia function as versatile sensory antennae regulating various cellular processes, but their role on compensatory beta-cell replication has not been examined. Here, we identify a significant enrichment of downregulated, cilia-annotated genes in pancreatic islets of diabetes-prone NZO mice as compared with diabetes-resistant B6-ob/ob mice. Among 327 differentially expressed mouse cilia genes, 81 human orthologs are also affected in islets of diabetic donors. Islets of nondiabetic mice and humans show a substantial overlap of upregulated cilia genes that are linked to cell-cycle progression. The shRNA-mediated suppression of KIF3A, essential for ciliogenesis, impairs division of MIN6 beta cells as well as in dispersed primary mouse and human islet cells, as shown by decreased BrdU incorporation. These findings demonstrate the substantial role of cilia-gene regulation on islet function and T2D risk.
Asunto(s)
Cilios/genética , Diabetes Mellitus Tipo 2/genética , Células Secretoras de Insulina/metabolismo , Transcriptoma , Animales , Ciclo Celular/genética , Línea Celular Tumoral , Células Cultivadas , Cilios/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Masculino , RatonesRESUMEN
BACKGROUND: Dietary intake of saturated fat is a likely contributor to nonalcoholic fatty liver disease (NAFLD) and insulin resistance, but the mechanisms that initiate these abnormalities in humans remain unclear. We examined the effects of a single oral saturated fat load on insulin sensitivity, hepatic glucose metabolism, and lipid metabolism in humans. Similarly, initiating mechanisms were examined after an equivalent challenge in mice. METHODS: Fourteen lean, healthy individuals randomly received either palm oil (PO) or vehicle (VCL). Hepatic metabolism was analyzed using in vivo 13C/31P/1H and ex vivo 2H magnetic resonance spectroscopy before and during hyperinsulinemic-euglycemic clamps with isotope dilution. Mice underwent identical clamp procedures and hepatic transcriptome analyses. RESULTS: PO administration decreased whole-body, hepatic, and adipose tissue insulin sensitivity by 25%, 15%, and 34%, respectively. Hepatic triglyceride and ATP content rose by 35% and 16%, respectively. Hepatic gluconeogenesis increased by 70%, and net glycogenolysis declined by 20%. Mouse transcriptomics revealed that PO differentially regulates predicted upstream regulators and pathways, including LPS, members of the TLR and PPAR families, NF-κB, and TNF-related weak inducer of apoptosis (TWEAK). CONCLUSION: Saturated fat ingestion rapidly increases hepatic lipid storage, energy metabolism, and insulin resistance. This is accompanied by regulation of hepatic gene expression and signaling that may contribute to development of NAFLD.REGISTRATION. ClinicalTrials.gov NCT01736202. FUNDING: Germany: Ministry of Innovation, Science, and Research North Rhine-Westfalia, German Federal Ministry of Health, Federal Ministry of Education and Research, German Center for Diabetes Research, German Research Foundation, and German Diabetes Association. Portugal: Portuguese Foundation for Science and Technology, FEDER - European Regional Development Fund, Portuguese Foundation for Science and Technology, and Rede Nacional de Ressonância Magnética Nuclear.
Asunto(s)
Tejido Adiposo/metabolismo , Grasas de la Dieta/efectos adversos , Metabolismo Energético/efectos de los fármacos , Resistencia a la Insulina , Hígado/metabolismo , Aceites de Plantas/efectos adversos , Tejido Adiposo/patología , Adulto , Animales , Citocina TWEAK , Grasas de la Dieta/administración & dosificación , Humanos , Hígado/patología , Masculino , Ratones , FN-kappa B/metabolismo , Enfermedad del Hígado Graso no Alcohólico/inducido químicamente , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Aceite de Palma , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Aceites de Plantas/administración & dosificación , Factores de Necrosis Tumoral/metabolismoRESUMEN
Combined use of metformin and a sodium glucose cotransporter 2 inhibitor (SGLT2I) is a promising treatment strategy for type 2 diabetes. The mechanism by which combination treatment provides better glycemic control than metformin or SGLT2I monotherapy remains elusive. Therefore, we investigated the physiological mechanism by which both compounds lower blood glucose concentrations in diabetic mice. We compared the potential of metformin and the SGLT2I AVE2268 alone or in combination to mitigate hyperglycemia and modulate glucose fluxes in db/db and diabetic Tallyho/JngJ mice. SGLT2I treatment alone elicited a rapid decline in circulating blood glucose levels, which appeared to induce endogenous glucose production. Supplementation of metformin dampened this counterresponse, and therefore, combination therapy more efficiently maintained glycemic control. Finally, combination treatment blunted postprandial glucose excursions and improved HbA1c levels within 2 weeks. We conclude that coapplication of metformin enhances the glucose-lowering actions of SGLT2I by restraining endogenous glucose production, which may provide long-term improvement of glycemic control in type 2 diabetic patients.
Asunto(s)
Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Glucosa/biosíntesis , Glucósidos/farmacología , Metformina/farmacología , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Animales , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animales de Enfermedad , Quimioterapia Combinada , Técnica de Clampeo de la Glucosa , Hemoglobina Glucada/metabolismo , Hiperglucemia/tratamiento farmacológico , Hiperglucemia/metabolismo , Hipoglucemiantes/farmacología , Ratones Noqueados , Ratones Obesos , Obesidad/metabolismo , Transportador 2 de Sodio-Glucosa/metabolismoRESUMEN
Genetic predisposition and environmental factors contribute to an individual's susceptibility to develop hepatosteatosis. In a systematic, comparative survey we focused on genotype-dependent and -independent adaptations early in the pathogenesis of hepatosteatosis by characterizing C3HeB/FeJ, C57BL/6NTac, C57BL/6J, and 129P2/OlaHsd mice after 7, 14, or 21 days high-fat-diet exposure. Strain-specific metabolic responses during diet challenge and liver transcript signatures in mild hepatosteatosis outline the suitability of particular strains for investigating the relationship between hepatocellular lipid content and inflammation, glucose homeostasis, insulin action, or organelle physiology. Genetic background-independent transcriptional adaptations in liver paralleling hepatosteatosis suggest an early increase in the organ's vulnerability to oxidative stress damage what could advance hepatosteatosis to steatohepatitis. "Universal" adaptations in transcript signatures and transcription factor regulation in liver link insulin resistance, type 2 diabetes mellitus, cancer, and thyroid hormone metabolism with hepatosteatosis, hence, facilitating the search for novel molecular mechanisms potentially implicated in the pathogenesis of human non-alcoholic-fatty-liver-disease.