Essential Role of Phosphoinositide 3-Kinase in Leptin-inducedK ATP Channel Activation in the Rat CRI-G1 Insulinoma Cell Line

Abstract

The mechanism by which leptin increases ATP-sensitive K ؉ (K ATP ) channel activity was investigated using the insulin-secreting cell line, CRI-G1. Wortmannin and LY 294002, inhibitors of phosphoinositide 3-kinase (PI3-kinase), prevented activation of K ATP channels by leptin. The inositol phospholipids phosphatidylinositol bisphosphate and phosphatidylinositol trisphosphate (PtdIns(3,4,5)P 3 ) mimicked the effect of leptin by increasing K ATP channel activity in whole-cell and insideout current recordings. LY 294002 prevented phosphatidylinositol bisphosphate, but not PtdIns(3,4,5)P 3 , from increasing K ATP channel activity, consistent with the latter lipid acting as a membrane-associated messenger linking leptin receptor activation and K ATP channels. Signaling cascades, activated downstream from PI 3-kinase, utilizing PtdIns(3,4,5)P 3 as a second messenger and commonly associated with insulin and cytokine action (MAPK, p70 ribosomal protein-S6 kinase, stress-activated protein kinase 2, p38 MAPK, and protein kinase B), do not appear to be involved in leptin-mediated activation of K ATP channels in this cell line. Although PtdIns(3,4,5)P 3 appears a plausible and attractive candidate for the messenger that couples K ATP channels to leptin receptor activation, direct measurement of PtdIns(3,4,5)P 3 demonstrated that insulin, but not leptin, increased global cellular levels of PtdIns(3,4,5)P 3 . Possible mechanisms to explain the involvement of PI 3-kinases in K ATP channel regulation are discussed.The hormone leptin, secreted by adipocytes, has a major influence on body weight homeostasis (1, 2). Although the hypothalamus is considered the main target for leptin, particularly with respect to body weight regulation, it is clear that this hormone has distinct actions on other peripheral, target organs. There have been several reports that leptin reduces insulin secretion from pancreatic beta cells (3-6), although this view is not shared by all investigators (7). One mechanism proposed to explain the leptin-induced reduction in insulin secretion is via activation of ATP-sensitive K ϩ (K ATP ) channels (8, 9). This increase in potassium current results in beta cell hyperpolarization, reduced calcium entry, and hence decreased insulin secretion. In addition, there are features common to both insulin-secreting cells and leptin-sensitive hypothalamic neurones (10, 11), most notably glucose responsiveness and the presence of K ATP channels, which are activated by exposure of the cells to leptin. The apparent involvement of both leptin receptors and K ATP channel activation in key systems involved in metabolic homeostasis has led us to examine the likely signal transduction pathways underlying this effect.The leptin receptor belongs to the class I cytokine receptor superfamily (1, 2), members of which are thought to signal via janus-tyrosine kinases. Activated janus-tyrosine kinases can mediate signaling via insulin receptor substrate proteins (12-14), which following tyrosine phosphorylation become docking sites for Sr...

Leptin-dependent Phosphorylation of PTEN Mediates Actin Restructuring and Activation of ATP-sensitive K+ Channels

“…Voltage clamp analysis (Fig. 4A) showed that leptin increased the mean cell slope conductance from 0.73 Ϯ 0.11 to 1.62 Ϯ 0.22 nanosiemens (n ϭ 19; p Ͻ 0.05), an action reduced by tolbutamide to 0.42 Ϯ 0.04 nanosiemens (n ϭ 14; p Ͻ 0.05), indicating that the increase in current responsible for the augmented slope conductance and ␤-cell hyperpolarization by leptin is due to K ATP channel opening (14,15,30). The presence of DMAT (10 M) in the electrode solution (and, therefore, cell interior) resulted in a mean resting membrane potential of Ϫ44.8 Ϯ 3.4 mV (p Ͼ 0.05 compared with control).…”

“…Instead, leptin inhibited the lipid and protein phosphatase, PTEN, which resulted in increased PtdIns(3,4,5)P 3 levels in the presence of active PI3K (12). Previously, PI3K-dependent leptin signaling had been shown to open ATP-sensitive (K ATP ) channels in rat hypothalamic neurons (13) and in rat and mouse insulin-secreting cells (12,14,15), resulting in cell hyperpolarization and inhibition of firing. K ATP activation by leptin is dependent on actin depolymerization in both cell types (12)(13)(14)(15).…”

“…Previously, PI3K-dependent leptin signaling had been shown to open ATP-sensitive (K ATP ) channels in rat hypothalamic neurons (13) and in rat and mouse insulin-secreting cells (12,14,15), resulting in cell hyperpolarization and inhibition of firing. K ATP activation by leptin is dependent on actin depolymerization in both cell types (12)(13)(14)(15). The connection between leptin-driven PI3K activity, actin re-modeling, and K ATP opening appears not to be due simply to increased PtdIns(3,4,5)P 3 but may also require coincident inhibition of PTEN protein and lipid phosphatase activity through increased PTEN phosphorylation (12).…”

Leptin and mTOR: Partners in metabolism and inflammation

“…18 Moreover, leptin has been shown to activate the PI3K in leukocytes, in a similar way to that observed for other members of the cytokine receptor superfamily. 16,17,[19][20][21][22] Leptin is a key intermediary between energy homeostasis and the immune system and may play roles in inflammation and obesity-related diseases including atherosclerosis and cancer. The characterization of leptin functions and signaling pathways in regulating lipid metabolism, inflammatory mediator production and lipid body biogenesis in macrophages and other cells are of importance for understanding the roles of leptin in the pathogenesis of inflammatory diseases including atherosclerosis.…”

Glucosensing neurons do more than just sense glucose

“…Transmitters or peptides which inhibit PLC or PI3-P through their actions on the GPCR would tend to oppose the actions of glucose or sulfonylureas. (Adapted from Harvey et al 27,28 and Baukrowitz and Fakler. 29 ) …”