RESUMEN
Leptin activates multiple signaling pathways in cells, including the phosphatidylinositol 3-kinase pathway, indicating a degree of cross-talk with insulin signaling. The exact mechanisms by which leptin alters this signaling pathway and how it relates to functional outputs are unclear at present. A previous study has established that leptin inhibits the activity of the phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10), an important tumor suppressor and modifier of phosphoinositide signaling. In this study we demonstrate that leptin phosphorylates multiple sites on the C-terminal tail of PTEN in hypothalamic and pancreatic beta-cells, an action not replicated by insulin. Inhibitors of the protein kinases CK2 and glycogen synthase kinase 3 (GSK3) block leptin-mediated PTEN phosphorylation. PTEN phosphorylation mutants reveal the critical role these sites play in transmission of the leptin signal to F-actin depolymerization. CK2 and GSK3 inhibitors also prevent leptin-mediated F-actin depolymerization and consequent ATP-sensitive K(+) channel opening. GSK3 kinase activity is inhibited by insulin but not leptin in hypothalamic cells. Both hormones increase N-terminal GSK3 serine phosphorylation, but in hypothalamic cells this action of leptin is transient. Leptin, not insulin, increases GSK3 tyrosine phosphorylation in both cell types. These results demonstrate a significant role for PTEN in leptin signal transmission and identify GSK3 as a potential important signaling node contributing to divergent outputs for these hormones.
Asunto(s)
Actinas/metabolismo , Adenosina Trifosfato/metabolismo , Leptina/farmacología , Fosfohidrolasa PTEN/metabolismo , Canales de Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Quinasa de la Caseína II/metabolismo , Línea Celular , Activación Enzimática , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutación/genética , Fosfohidrolasa PTEN/química , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacosRESUMEN
In obesity and diabetes, the ability of hypothalamic neurons to sense and transduce changes in leptin and insulin levels is compromised. The effects of both hormones require intracellular signalling via the PI3-kinase pathway, which is inhibited by the phosphatase PTEN. We show that leptin-stimulated F-actin depolymerization in mouse hypothalamic cells is inhibited by PTEN, a process involving independent effects of both its lipid and protein phosphatase activities. Potentially mediating this F-actin depolymerization, leptin, but not insulin, stimulated the phosphorylation of PTEN in a CK2 dependent manner, and inhibited its phosphatase activity. Similarly, hyperpolarization of mouse pancreatic beta-cells by leptin also requires coincident PtdIns(3,4,5)P3 generation and actin depolymerization, and could be inhibited by mechanisms requiring both the lipid and protein phosphatase activities of PTEN. These results demonstrate a critical role for PTEN in leptin signalling and indicate a mechanism by which leptin and insulin can produce PI3K dependent differential cellular outputs.
Asunto(s)
Hipotálamo/citología , Células Secretoras de Insulina/metabolismo , Leptina/metabolismo , Fosfohidrolasa PTEN/metabolismo , Transducción de Señal/fisiología , Actinas/metabolismo , Animales , Células Cultivadas , Hipotálamo/metabolismo , Células Secretoras de Insulina/citología , Ratones , Fosfohidrolasa PTEN/antagonistas & inhibidores , Fosfohidrolasa PTEN/genética , Técnicas de Placa-Clamp , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilación , Canales de Potasio/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de LeptinaRESUMEN
BACKGROUND: Leptin and insulin are long-term regulators of body weight. They act in hypothalamic centres to modulate the function of specific neuronal subtypes, by altering transcriptional control of releasable peptides and by modifying neuronal electrical activity. A key cellular signalling intermediate, implicated in control of food intake by these hormones, is the enzyme phosphoinositide 3-kinase. In this study we have explored further the linkage between this enzyme and other cellular mediators of leptin and insulin action on rat arcuate nucleus neurones and the mouse hypothalamic cell line, GT1-7. RESULTS: Leptin and insulin increased the levels of various phosphorylated signalling intermediates, associated with the JAK2-STAT3, MAPK and PI3K cascades in the arcuate nucleus. Inhibitors of PI3K were shown to reduce the hormone driven phosphorylation through the PI3K and MAPK pathways. Using isolated arcuate neurones, leptin and insulin were demonstrated to increase the activity of KATP channels in a PI3K dependent manner, and to increase levels of PtdIns(3,4,5)P3. KATP activation by these hormones in arcuate neurones was also sensitive to the presence of the actin filament stabilising toxin, jasplakinolide. Using confocal imaging of fluorescently labelled actin and direct analysis of G- and F-actin concentration in GT1-7 cells, leptin was demonstrated directly to induce a re-organization of cellular actin, by increasing levels of globular actin at the expense of filamentous actin in a PI3-kinase dependent manner. Leptin stimulated PI3-kinase activity in GT1-7 cells and an increase in PtdIns(3,4,5)P3 could be detected, which was prevented by PI3K inhibitors. CONCLUSIONS: Leptin and insulin mediated phosphorylation of cellular signalling intermediates and of KATP channel activation in arcuate neurones is sensitive to PI3K inhibition, thus strengthening further the likely importance of this enzyme in leptin and insulin mediated energy homeostasis control. The sensitivity of leptin and insulin stimulation of KATP channel opening in arcuate neurones to jasplakinolide indicates that cytoskeletal remodelling may be an important contributor to the cellular signalling mechanisms of these hormones in hypothalamic neurones. This hypothesis is reinforced by the finding that leptin induces actin filament depolymerization, in a PI3K dependent manner in a mouse hypothalamic cell line.
Asunto(s)
Citoesqueleto de Actina/ultraestructura , Núcleo Arqueado del Hipotálamo/fisiología , Insulina/farmacología , Leptina/farmacología , Neuronas/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Canales de Potasio/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Núcleo Arqueado del Hipotálamo/citología , Línea Celular , Células Cultivadas , Masculino , Ratones , Neuronas/enzimología , Neuronas/ultraestructura , Técnicas de Placa-Clamp , Fosforilación , Ratas , Ratas Sprague-Dawley , Transducción de SeñalRESUMEN
BACKGROUND: 3-guanidinopropionic acid derivatives reduce body weight in obese, diabetic mice. We have assessed whether one of these analogues, the aminoguanidine carboxylate BVT.12777, opens KATP channels in rat insulinoma cells, by the same mechanism as leptin. RESULTS: BVT.12777 hyperpolarized CRI-G1 rat insulinoma cells by activation of KATP channels. In contrast, BVT.12777 did not activate heterologously expressed pancreatic beta-cell KATP subunits directly. Although BVT.12777 stimulated phosphorylation of MAPK and STAT3, there was no effect on enzymes downstream of PI3K. Activation of KATP in CRI-G1 cells by BVT.12777 was not dependent on MAPK or PI3K activity. Confocal imaging showed that BVT.12777 induced a re-organization of cellular actin. Furthermore, the activation of KATP by BVT.12777 in CRI-G1 cells was demonstrated to be dependent on actin cytoskeletal dynamics, similar to that observed for leptin. CONCLUSIONS: This study shows that BVT.12777, like leptin, activates KATP channels in insulinoma cells. Unlike leptin, BVT.12777 activates KATP channels in a PI3K-independent manner, but, like leptin, channel activation is dependent on actin cytoskeleton remodelling. Thus, BVT.12777 appears to act as a leptin mimetic, at least with respect to KATP channel activation, and may bypass up-stream signalling components of the leptin pathway.