Inhibitory Effects of Leptin on Pancreatic α-Cell Function

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)OBJECTIVE-Leptin released from adipocytes plays a key role in the control of food intake, energy balance, and glucose homeostasis. In addition to its central action, leptin directly affects pancreatic beta-cells, inhibiting insulin secretion, and, thus, modulating glucose homeostasis. However, despite the importance of glucagon secretion in glucose homeostasis, the role of leptin in a-cell function has not been studied in detail. In the present study, we have investigated this functional interaction. RESEARCH DESIGN AND METHODS-The presence of leptin receptors (ObR) was demonstrated by RT-PCR analysis, Western blot, and immunocytochemistry. Electrical activity was analyzed by patch-clamp and Ca(2+) signals by confocal microscopy. Exocytosis and glucagon secretion were assessed using fluorescence methods and radioimmunoassay, respectively. RESULTS-The expression of several ObR isoforms (a-e) was detected in glucagon-secreting alpha TC1-9 cells. ObRb, the main isoform involved in leptin signaling, was identified at the protein level in alpha TC1-9 cells as well as in mouse and human alpha-cells. The application of leptin (6.25 nmol/l) hyperpolarized the alpha-cell membrane potential, suppressing the electrical activity induced by 0.5 mmol/l glucose. Additionally, leptin inhibited Ca(2+) signaling in alpha TC1-9 cells and in mouse and human alpha-cells within intact islets. A similar result occurred with 0.625 nmol/l leptin. These effects were accompanied by a decrease in glucagon secretion from mouse islets and were counteracted by the phosphatidylinositol 3-kinase inhibitor, wortmannin, suggesting the involvement of this pathway in leptin action. CONCLUSIONS-These results demonstrate that leptin inhibits alpha-cell function, and, thus, these cells are involved in the adipo-insular communication. Diabetes 58:1616-1624, 200958716161624Ministerio de Educacion y Ciencia [BFU2007-67607, PCI2005-A7-0131, BFU2008-01492, SAF2006-07382]Ministerio de Ciencia a InnovacionFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Ministerio de Educacion y Ciencia [BFU2007-67607, PCI2005-A7-0131, BFU2008-01492, SAF2006-07382]FAPESP [2008/53811-8

α1A-Adrenergic Receptor Induces Activation of Extracellular Signal-Regulated Kinase 1/2 through Endocytic Pathway

G protein-coupled receptors (GPCRs) activate mitogen-activated protein kinases through a number of distinct pathways in cells. Increasing evidence has suggested that endosomal signaling has an important role in receptor signal transduction. Here we investigated the involvement of endocytosis in α1A-adrenergic receptor (α1A-AR)-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Agonist-mediated endocytic traffic of α1A-AR was assessed by real-time imaging of living, stably transfected human embryonic kidney 293A cells (HEK-293A). α1A-AR was internalized dynamically in cells with agonist stimulation, and actin filaments regulated the initial trafficking of α1A-AR. α1A-AR-induced activation of ERK1/2 but not p38 MAPK was sensitive to disruption of endocytosis, as demonstrated by 4°C chilling, dynamin mutation and treatment with cytochalasin D (actin depolymerizing agent). Activation of protein kinase C (PKC) and C-Raf by α1A-AR was not affected by 4°C chilling or cytochalasin D treatment. U73122 (a phospholipase C [PLC] inhibitor) and Ro 31–8220 (a PKC inhibitor) inhibited α1B-AR- but not α1A-AR-induced ERK1/2 activation. These data suggest that the endocytic pathway is involved in α1A-AR-induced ERK1/2 activation, which is independent of Gq/PLC/PKC signaling

Pulsatility of insulin release – a clinically important phenomenon

The mechanisms and clinical importance of pulsatile insulin release are presented against the background of more than half a century of companionship with the islets of Langerhans. The insulin-secreting β-cells are oscillators with intrinsic variations of cytoplasmic ATP and Ca2+. Within the islets the β-cells are mutually entrained into a common rhythm by gap junctions and diffusible factors (ATP). Synchronization of the different islets in the pancreas is supposed to be due to adjustment of the oscillations to the same phase by neural output of acetylcholine and ATP. Studies of hormone secretion from the perfused pancreas of rats and mice revealed that glucose induces pulses of glucagon anti-synchronous with pulses of insulin and somatostatin. The anti-synchrony may result from a paracrine action of somatostatin on the glucagon-producing α-cells. Purinoceptors have a key function for pulsatile release of islet hormones. It was possible to remove the glucagon and somatostatin pulses with maintenance of those of insulin with an inhibitor of the P2Y1 receptors. Knock-out of the adenosine A1 receptor prolonged the pulses of glucagon and somatostatin without affecting the duration of the insulin pulses. Studies of isolated human islets indicate similar relations between pulses of insulin, glucagon, and somatostatin as found during perfusion of the rodent pancreas. The observation of reversed cycles of insulin and glucagon adds to the understanding how the islets regulate hepatic glucose production. Current protocols for pulsatile intravenous infusion therapy (PIVIT) should be modified to mimic the anti-synchrony between insulin and glucagon normally seen in the portal blood

Stimulation of insulin release by isosmolar addition of permeant molecules

Pancreatic beta-cells are known to respond to hyposmolar stress by releasing insulin. It was evident from perifusion studies using islet cells from ob/ob-mice mixed with polyacrylamide beads that a similar type of secretory response can be obtained by isosmolar addition of 10-25 mM of the rapidly penetrating urea molecule. There was no effect with hyperosmolar addition of urea. The urea-induced insulin release differed from the ordinary stimulation of secretion in not disappearing but being more pronounced after previous heating to 45 degrees C or removal of extracellular Ca2+. Isosmolar urea was exceptional as an insulin secretagogue in being effective also in the presence of the alpha 2-adrenergic agonist clonidine or when lowering the temperature to 24 degrees C. Further support for the idea that isosmolar addition of rapidly penetrating molecules induces insulin release was obtained by testing non-metabolizable glucose analogues. Whereas 25 mM 3-O-methyl-D-glucose doubled the secretory rate within 4 min, the non-permeant L-glucose had only a slight initial action. When not compensating for the alterations of the medium osmolarity 3-O-methyl-D-glucose was without effect. Although expansion of beta-cells cannot explain the existence of a pronounced initial secretory response to D-glucose it may under certain conditions contribute to the stimulatory effects of the sugar.</p