RESUMEN
Endothelial cells line the blood and lymphatic vasculature, and act as an essential physical barrier, control nutrient transport, facilitate tissue immunosurveillance and coordinate angiogenesis and lymphangiogenesis1,2. In the intestine, dietary and microbial cues are particularly important in the regulation of organ homeostasis. However, whether enteric endothelial cells actively sense and integrate such signals is currently unknown. Here we show that the aryl hydrocarbon receptor (AHR) acts as a critical node for endothelial cell sensing of dietary metabolites in adult mice and human primary endothelial cells. We first established a comprehensive single-cell endothelial atlas of the mouse small intestine, uncovering the cellular complexity and functional heterogeneity of blood and lymphatic endothelial cells. Analyses of AHR-mediated responses at single-cell resolution identified tissue-protective transcriptional signatures and regulatory networks promoting cellular quiescence and vascular normalcy at steady state. Endothelial AHR deficiency in adult mice resulted in dysregulated inflammatory responses and the initiation of proliferative pathways. Furthermore, endothelial sensing of dietary AHR ligands was required for optimal protection against enteric infection. In human endothelial cells, AHR signalling promoted quiescence and restrained activation by inflammatory mediators. Together, our data provide a comprehensive dissection of the effect of environmental sensing across the spectrum of enteric endothelia, demonstrating that endothelial AHR signalling integrates dietary cues to maintain tissue homeostasis by promoting endothelial cell quiescence and vascular normalcy.
Asunto(s)
Células Endoteliales , Receptores de Hidrocarburo de Aril , Humanos , Animales , Ratones , Receptores de Hidrocarburo de Aril/metabolismo , Células Endoteliales/metabolismo , Intestinos , Transducción de Señal , Homeostasis , LigandosRESUMEN
AIMS/HYPOTHESIS: Hypoglycaemia is a major barrier to good glucose control in type 1 diabetes. Frequent hypoglycaemic episodes impair awareness of subsequent hypoglycaemic bouts. Neural changes underpinning awareness of hypoglycaemia are poorly defined and molecular mechanisms by which glial cells contribute to hypoglycaemia sensing and glucose counterregulation require further investigation. The aim of the current study was to examine whether, and by what mechanism, human primary astrocyte (HPA) function was altered by acute and recurrent low glucose (RLG). METHODS: To test whether glia, specifically astrocytes, could detect changes in glucose, we utilised HPA and U373 astrocytoma cells and exposed them to RLG in vitro. This allowed measurement, with high specificity and sensitivity, of RLG-associated changes in cellular metabolism. We examined changes in protein phosphorylation/expression using western blotting. Metabolic function was assessed using a Seahorse extracellular flux analyser. Immunofluorescent imaging was used to examine cell morphology and enzymatic assays were used to measure lactate release, glycogen content, intracellular ATP and nucleotide ratios. RESULTS: AMP-activated protein kinase (AMPK) was activated over a pathophysiologically relevant glucose concentration range. RLG produced an increased dependency on fatty acid oxidation for basal mitochondrial metabolism and exhibited hallmarks of mitochondrial stress, including increased proton leak and reduced coupling efficiency. Relative to glucose availability, lactate release increased during low glucose but this was not modified by RLG. Basal glucose uptake was not modified by RLG and glycogen levels were similar in control and RLG-treated cells. Mitochondrial adaptations to RLG were partially recovered by maintaining euglycaemic levels of glucose following RLG exposure. CONCLUSIONS/INTERPRETATION: Taken together, these data indicate that HPA mitochondria are altered following RLG, with a metabolic switch towards increased fatty acid oxidation, suggesting glial adaptations to RLG involve altered mitochondrial metabolism that could contribute to defective glucose counterregulation to hypoglycaemia in diabetes.
Asunto(s)
Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Ácidos Grasos/metabolismo , Glucosa/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Adolescente , Línea Celular , Células Cultivadas , Humanos , Hipoglucemia/metabolismo , Immunoblotting , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oxidación-Reducción/efectos de los fármacosRESUMEN
AIM: To test the hypothesis that, given the role of AMP-activated protein kinase (AMPK) in regulating intracellular ATP levels, AMPK may alter ATP release from astrocytes, the main sources of extracellular ATP (eATP) within the brain. MATERIALS AND METHODS: Measurements of ATP release were made from human U373 astrocytoma cells, primary mouse hypothalamic (HTAS) and cortical astrocytes (CRTAS) and wild-type and AMPK α1/α2 null mouse embryonic fibroblasts (MEFs). Cells were treated with drugs known to modulate AMPK activity: A-769662, AICAR and metformin, for up to 3 hours. Intracellular calcium was measured using Fluo4 and Fura-2 calcium-sensitive fluorescent dyes. RESULTS: In U373 cells, A-769662 (100 µM) increased AMPK phosphorylation, whereas AICAR and metformin (1 mM) induced a modest increase or had no effect, respectively. Only A-769662 increased eATP levels, and this was partially blocked by AMPK inhibitor Compound C. A-769662-induced increases in eATP were preserved in AMPK α1/α2 null MEF cells. A-769662 increased intracellular calcium in U373, HTAS and CRTAS cells and chelation of intracellular calcium using BAPTA-AM reduced A-769662-induced eATP levels. A-769662 also increased ATP release from a number of other central and peripheral endocrine cell types. CONCLUSIONS: AMPK is required to maintain basal eATP levels but is not required for A-769662-induced increases in eATP. A-769662 (>50 µM) enhanced intracellular calcium levels leading to ATP release in an AMPK and purinergic receptor independent pathway.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Trifosfato/metabolismo , Astrocitos/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Activadores de Enzimas/farmacología , Hipoglucemiantes/farmacología , Pironas/farmacología , Tiofenos/farmacología , Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/genética , Acetil-CoA Carboxilasa/química , Acetil-CoA Carboxilasa/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/citología , Astrocitos/metabolismo , Compuestos de Bifenilo , Línea Celular , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Embrión de Mamíferos/citología , Activación Enzimática/efectos de los fármacos , Humanos , Hipotálamo/citología , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/agonistas , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacosRESUMEN
The cardiac phosphoprotein phospholemman (PLM) regulates the cardiac sodium pump, activating the pump when phosphorylated and inhibiting it when palmitoylated. Protein palmitoylation, the reversible attachment of a 16 carbon fatty acid to a cysteine thiol, is catalyzed by the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases. The cell surface palmitoyl acyltransferase DHHC5 regulates a growing number of cellular processes, but relatively few DHHC5 substrates have been identified to date. We examined the expression of DHHC isoforms in ventricular muscle and report that DHHC5 is among the most abundantly expressed DHHCs in the heart and localizes to caveolin-enriched cell surface microdomains. DHHC5 coimmunoprecipitates with PLM in ventricular myocytes and transiently transfected cells. Overexpression and silencing experiments indicate that DHHC5 palmitoylates PLM at two juxtamembrane cysteines, C40 and C42, although C40 is the principal palmitoylation site. PLM interaction with and palmitoylation by DHHC5 is independent of the DHHC5 PSD-95/Discs-large/ZO-1 homology (PDZ) binding motif, but requires a â¼ 120 amino acid region of the DHHC5 intracellular C-tail immediately after the fourth transmembrane domain. PLM C42A but not PLM C40A inhibits the Na pump, indicating PLM palmitoylation at C40 but not C42 is required for PLM-mediated inhibition of pump activity. In conclusion, we demonstrate an enzyme-substrate relationship for DHHC5 and PLM and describe a means of substrate recruitment not hitherto described for this acyltransferase. We propose that PLM palmitoylation by DHHC5 promotes phospholipid interactions that inhibit the Na pump.
Asunto(s)
Proteínas de la Membrana/química , Proteínas de la Membrana/fisiología , Fosfoproteínas/química , Aciltransferasas , Secuencias de Aminoácidos , Animales , Membrana Celular/enzimología , Perros , Endocitosis , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Lipoilación , Ratones , Miocardio/metabolismo , Plasticidad Neuronal , Fosfolípidos/química , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , ARN Interferente Pequeño/metabolismo , Ratas , Sodio/química , Especificidad por Sustrato , SinapsisRESUMEN
Aim: We evaluated the efficacy of a novel brain permeable "metformin-like" AMP-activated protein kinase activator, R481, in regulating glucose homeostasis. Materials and Methods: We used glucose sensing hypothalamic GT1-7 neuronal cells and pancreatic αTC1.9 α-cells to examine the effect of R481 on AMPK pathway activation and cellular metabolism. Glucose tolerance tests and hyperinsulinemic-euglycemic and hypoglycemic clamps were used in Sprague-Dawley rats to assess insulin sensitivity and hypoglycemia counterregulation, respectively. Results: In vitro, we demonstrate that R481 increased AMPK phosphorylation in GT1-7 and αTC1.9 cells. In Sprague-Dawley rats, R481 increased peak glucose levels during a glucose tolerance test, without altering insulin levels or glucose clearance. The effect of R481 to raise peak glucose levels was attenuated by allosteric brain permeable AMPK inhibitor SBI-0206965. This effect was also completely abolished by blockade of the autonomic nervous system using hexamethonium. During hypoglycemic clamp studies, R481 treated animals had a significantly lower glucose infusion rate compared to vehicle treated controls. Peak plasma glucagon levels were significantly higher in R481 treated rats with no change to plasma adrenaline levels. In vitro, R481 did not alter glucagon release from αTC1.9 cells, but increased glycolysis. Non brain permeable AMPK activator R419 enhanced AMPK activity in vitro in neuronal cells but did not alter glucose excursion in vivo. Conclusions: These data demonstrate that peripheral administration of the brain permeable "metformin-like" AMPK activator R481 increases blood glucose by activation of the autonomic nervous system and amplifies the glucagon response to hypoglycemia in rats. Taken together, our data suggest that R481 amplifies the counterregulatory response to hypoglycemia by a central rather than a direct effect on the pancreatic α-cell. These data provide proof-of-concept that central AMPK could be a target for future drug development for prevention of hypoglycemia in diabetes.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Sistema Nervioso Autónomo/efectos de los fármacos , Glucemia/efectos de los fármacos , Hipoglucemia/metabolismo , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/efectos de los fármacos , Animales , Sistema Nervioso Autónomo/fisiología , Benzamidas/farmacología , Glucemia/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Células Cultivadas , Hipoglucemia/patología , Hipoglucemia/fisiopatología , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Masculino , Permeabilidad/efectos de los fármacos , Piperidinas/farmacología , Pirimidinas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
The unsaturated fatty acid, oleate exhibits anorexigenic properties reducing food intake and hepatic glucose output. However, its mechanism of action in the hypothalamus has not been fully determined. This study investigated the effects of oleate and glucose on GT1-7 mouse hypothalamic cells (a model of glucose-excited (GE) neurons) and mouse arcuate nucleus (ARC) neurons. Whole-cell and perforated patch-clamp recordings, immunoblotting and cell energy status measures were used to investigate oleate- and glucose-sensing properties of mouse hypothalamic neurons. Oleate or lowered glucose concentration caused hyperpolarization and inhibition of firing of GT1-7 cells by the activation of ATP-sensitive K+ channels (KATP). This effect of oleate was not dependent on fatty acid oxidation or raised AMP-activated protein kinase activity or prevented by the presence of the UCP2 inhibitor genipin. Oleate did not alter intracellular calcium, indicating that CD36/fatty acid translocase may not play a role. However, oleate activation of KATP may require ATP metabolism. The short-chain fatty acid octanoate was unable to replicate the actions of oleate on GT1-7 cells. Although oleate decreased GT1-7 cell mitochondrial membrane potential there was no change in total cellular ATP or ATP/ADP ratios. Perforated patch and whole-cell recordings from mouse hypothalamic slices demonstrated that oleate hyperpolarized a subpopulation of ARC GE neurons by KATP activation. Additionally, in a separate small population of ARC neurons, oleate application or lowered glucose concentration caused membrane depolarization. In conclusion, oleate induces KATP-dependent hyperpolarization and inhibition of firing of a subgroup of GE hypothalamic neurons without altering cellular energy charge.