22 research outputs found
The cannabinoid ligand LH-21 reduces anxiety and improves glucose handling in diet-induced obese pre-diabetic mice
LH-21 is a triazol derivative that has been described as a low-permeant neutral CB1 antagonist, though its pharmacology is still unclear. It has been associated with anti-obesity actions in obese rats. However, its role in preventing type 2 diabetes (T2D) onset have not been studied yet. Given CB1 receptors remain as potential pharmacological targets to fight against obesity and T2D, we wanted to explore the metabolic impact of this compound in an animal model of obesity and pre-diabetes as well as the lack of relevant actions in related central processes such as anxiety. C57BL/6J mice were rendered obese and pre-diabetic by feeding a high-fat diet for 15 weeks and then treated with LH-21 or vehicle for two weeks. Food intake, body weight and glucose handling were assessed, together with other relevant parameters. Behavioural performance was evaluated by the open field test and the elevated plus maze. LH-21 did not affect food intake nor body weight but it improved glucose handling, displaying tissue-specific beneficial actions. Unexpectedly, LH-21 induced anxiolysis and reverted obesity-induced anxiety, apparently through GPR55 receptor. These results suggest that LH-21 can be a new candidate to fight against diabetes onset. Indeed, this compound shows potential in counteracting obesity-related anxiety.España, Ministerio de Sanidad 13/00309 to F.J.B.S. and PI13/00593 to B.R.GConsejería de Salud Junta de Andalucía C-0070-201
The diet-derived short chain fatty acid propionate improves beta-cell function in humans and stimulates insulin secretion from human islets in vitro
Aims:
Diet-derived short chain fatty acids (SCFAs) improve glucose homeostasis in vivo, but the role of individual SCFAs and their mechanisms of action have not been defined. This study evaluated the effects of increasing colonic delivery of the SCFA propionate on β-cell function in humans and the direct effects of propionate on isolated human islets in vitro.
Materials and Methods:
For 24 weeks human subjects ingested an inulin-propionate ester that delivers propionate to the colon. Acute insulin, GLP-1 and non-esterified fatty acid (NEFA) levels were quantified pre- and post-supplementation in response to a mixed meal test. Expression of the SCFA receptor FFAR2 in human islets was determined by western blotting and immunohistochemistry. Dynamic insulin secretion from perifused human islets was quantified by radioimmunoassay and islet apoptosis was determined by quantification of caspase 3/7 activities.
Results:
Colonic propionate delivery in vivo was associated with improved β-cell function with increased insulin secretion that was independent of changes in GLP-1 levels. Human islet β-cells expressed FFAR2 and propionate potentiated dynamic glucose-stimulated insulin secretion in vitro, an effect that was dependent on signalling via protein kinase C. Propionate also protected human islets from apoptosis induced by the NEFA sodium palmitate and inflammatory cytokines.
Conclusions:
Our results indicate that propionate has beneficial effects on β-cell function in vivo, and in vitro analyses demonstrated that it has direct effects to potentiate glucose-stimulated insulin release and maintain β-cell mass through inhibition of apoptosis. These observations support ingestion of propiogenic dietary fibres to maintain healthy glucose homeostasis
The diet-derived short chain fatty acid propionate improves beta-cell function in humans and stimulates insulin secretion from human islets in vitro
Los receptores CB1 y GPR55 en la fisiología del islote pancreático y en la fisiopatología de la diabetes tipo 2
A continuación, resumimos los resultados más significativos del trabajo realizado: i)Los
CB1 modulan la actividad de las proteínas AKT, CREB y AMPK en islotes de ratones
sanos y humanos, sugiriendo que podrían jugar un papel en la proliferación e
incrementos de masa de los mismos. Esta modulación está disminuida en islotes de
animales pre-diabéticos pudiendo colaborar al desarrollo de T2D. ii)Los receptores
GPR55 manifiestan efectos especie-dependientes al modular AKT, CREB y AMPK en
islotes de ratones sanos pero sólo AKT en humanos, proteína que podría ser
responsable de los efectos biológicos de su ligando endógeno. iii)Los CB1 y GPR55
modulan la GSIS y la [Ca2+]i en islotes de ratones sanos y de humanos. A
concentraciones bajas, LH-21 incrementa la GSIS y la [Ca2+]i modulando a GPR55 y no a
CB1 como podría pensarse. De manera similar, Abn-CBD, potencia la secreción de
insulina a través de mecanismos dependientes e independientes de GPR55. iv)Los CB1
y la vía de señalización de mTORC1 están funcionalmente conectados, modulando la
GSIS en islotes de ratones sanos. La conexión CB1-mTOR desaparece en islotes de
ratones pre-diabéticos, lo que sugiere que ésta contribuye a la fisiopatología de T2D.
v)Los CB1, a baja glucosa, modulan el contenido de ROS en islotes de ratones sanos.
Dicha modulación está alterada en islotes de ratones pre-diabéticos sugiriendo,
también, que afecta al desarrollo de T2D. vi)Finalmente, LH-21 y Abn-CBD, de manera
dependiente de GPR55, protegen de la apoptosis a los islotes de ratón y humanos,
pudiendo así ser útiles para evitar la muerte celular producida durante T2D.La diabetes tipo 2 (T2D), es una enfermedad caracterizada por hiperglucemia, debida a
una combinación entre resistencia a la insulina y una progresiva disfunción y pérdida
de masa de las células β que impide compensarla liberando mayor insulina. La
secreción de insulina estimulada por glucosa (GSIS) está modulada por la propia
insulina y otros factores como los endocannabinoides. Estas sustancias, ejercen sus
funciones reguladoras en las células β actuando principalmente sobre receptores
cannabinoides CB1 (CB1) y otros relacionados como los GPR55.
En esta tesis, hemos estudiado el papel modulador que CB1 y GPR55 ejercen en islotes
pancreáticos (humanos, y de ratones sanos, pre-diabéticos (obtenidos tras el
suministro prolongado de una dieta rica en grasa) y GPR55-/-), sobre las rutas de
señalización de AKT, AMPK y CREB, que controlan la proliferación, supervivencia y
masa de las células β, y que suelen alterarse en T2D. Para ello, los islotes se trataron
con rimonabant ó LH-21, antagonistas de CB1, ó Abn-CBD, agonista de GPR55. Se han
estudiado también los efectos moduladores de estos receptores sobre la GSIS y la
[Ca2+]i tras tratamiento farmacológico, y cómo el antagonismo de CB1 afecta a la ruta
de señalización de mTOR en islotes de ratones sanos y pre-diabéticos, y su efecto
sobre la GSIS. Asimismo, se ha investigado el papel de CB1 en la generación de ROS en
islotes de rata, ratones sanos y pre-diabéticos, y el jugado por GPR55 en la apoptosis
en islotes de ratones sanos, GPR55-/-
y de humanos
Identifying signalling pathways regulated by GPRC5B in β-cells by CRISPR-Cas9-mediated genome editing
Background/Aims: CRISPR-Cas9, a RNA-guided targeted genome editing tool, has revolutionized genetic engineering by offering the ability to precisely modify DNA. GPRC5B is an orphan receptor belonging to the group C family of G protein-coupled receptors (GPCRs). In this study, we analysed the functional roles of the Gprc5b receptor in MIN6 β-cells using CRISPR-Cas9 and transient over-expression of Gprc5b. Methods: The optimal transfection reagent for use in MIN6 β-cells was determined by analysing efficiency of GFP plasmid delivery by cell sorting. A MIN6 β-cell line in which Gprc5b expression was knocked down (Gprc5b KD) was generated using CRISPR-Cas9 technology. Gprc5b receptor mRNA expression, proliferation, apoptosis, Cignal 45-Pathway Reporter Array signalling and western blot assays were carried out using Gpcr5b KD MIN6 β-cells that had been transiently transfected with different concentrations of mouse Gprc5b plasmid to over-express Gprc5b. Results: JetPRIME® was the best candidate for MIN6 β-cell transfection, providing approximately 30% transfection efficiency. CRISPR-Cas9 technology targeting Gprc5b led to stable knock-down of this receptor in MIN6 β-cells and its re-expression induced proliferation and potentiated cytokine- and palmitate-induced apoptosis. The Cignal 45 Reporter analysis indicated Gprc5b-dependent regulation of apoptotic and proliferative pathways, and western blotting confirmed activation of signalling via TGF-β and IFNγ. Conclusion: This study provides evidence of CRISPR-Cas9 technology being used to down-regulate Gprc5b expression in MIN6 β-cells. This strategy allowed us to identify signalling pathways linking GPRC5B receptor expression to β-cell proliferation and apoptosis
Dynamic Profiling of Insulin Secretion and ATP Generation in Isolated Human and Mouse Islets Reveals Differential Glucose Sensitivity
Background/Aims: Rodent islets are often used for basic science research but they do not always recapitulate signalling events in human islets. This study evaluated the glucose-dependent responses of human and mouse islets in terms of dynamic insulin secretion, metabolic coupling and the role of glucose transporters. Methods: Glucose-induced insulin secretion from isolated mouse and human islets was profiled by perifusion and islet ATP levels were measured by chemoluminescence assay. Glucose transporter expression was determined by qPCR and western blotting. Results: Human islets show a left-shifted glucose concentration-insulin secretion profile compared to mouse islets. These data are consistent with glucose transporter expression, with human islets expressing mainly GLUT1 and GLUT3, and GLUT2 being the predominant transporter in mouse islets. Using the GLUT1 inhibitor STF-31 we unveiled an important role for GLUT1 for differences in glucose-induced insulin secretion profiles observed between the two species. Conclusion: The high affinity of GLUT1/3 for glucose reflects the left-shifted glucose-induced insulin secretory response of human islets and the impairment of insulin secretion from human islets after STF-31 treatment indicates an important role for GLUT1 in human islet stimulus-secretion coupling. Our data provide further insight into key differences between insulin secretion regulation in mouse and human islets