Leptin modulates pancreatic β-cell membrane potential through Src kinase–mediated phosphorylation of NMDA receptors

Abstract

The adipocyte-derived hormone leptin increases trafficking of KATP and Kv2.1 channels to the pancreatic β-cell surface, resulting in membrane hyperpolarization and suppression of insulin secretion. We have previously shown that this effect of leptin is mediated by the NMDA subtype of glutamate receptors (NMDARs). It does so by potentiating NMDAR activity, thus enhancing Ca2+ influx and the ensuing downstream signaling events that drive channel trafficking to the cell surface. However, the molecular mechanism by which leptin potentiates NMDARs in β-cells remains unknown. Here we report that leptin augments NMDAR function via Src kinase-mediated phosphorylation of the GluN2A subunit. Leptin-induced membrane hyperpolarization diminished upon pharmacological inhibition of GluN2A but not GluN2B, indicating involvement of GluN2A-containing NMDARs. GluN2A harbors tyrosine residues which when phosphorylated by Src family kinases potentiate NMDAR activity. We found that leptin increases phosphorylation of Y418 in Src, an indicator of kinase activation. Pharmacological inhibition of Src or overexpression of a kinase-dead Src mutant prevented the effect of leptin, while a Src kinase activator peptide mimicked it. Using mutant GluN2A overexpression, we show that Y1292 and Y1387 but not Y1325 are responsible for the effect of leptin. Importantly, β-cells from db/db mice, a type 2 diabetes mouse model lacking functional leptin receptors, or from obese diabetic human donors failed to respond to leptin but hyperpolarized in response to NMDA. Our study reveals a signaling pathway wherein leptin modulates NMDARs via Src to regulate β-cell excitability and suggests NMDARs as a potential target to overcome leptin resistance.

AKAP79/150 coordinates leptin-induced PKA activation to regulate KATPchannel trafficking in pancreatic β-cells

“…However, this effect of leptin was greatly attenuated by the presence of st-Ht31 indicating that blocking PKA from binding AKAPs prohibits leptin from increasing KATP channel surface density. Previously we have shown that the increased abundance of KATP channels in the membrane enhances total K + conductance and causes -cells to hyperpolarize [7][8][9][10] . To further verify the importance of PKA-AKAP interactions for KATP channel translocation we monitored the effects of st-Ht31 on cell membrane potential following leptin treatment using cell-attached current clamp recording, which is a non-invasive approach for detecting changes in membrane potential without disturbing cellular integrity or compromising intracellular soluble factors important for signaling 34,35 .…”

“…We have previously shown that leptin promotes trafficking of KATP channels to the plasma membrane, causing increased K + conductance and cell hyperpolarization in rat insulinoma INS-1 832/13 cells as well as primary mouse and human -cells [7][8][9][10] . Our prior work revealed that a critical event for this process is actin remodeling 7 , which presumably allows vesicles containing potassium channels to translocate and insert into the -cell membrane [26][27][28][29] .…”

“…Dissociation of human pancreatic -cells. Human β-cells were dissociated from human islets obtained through the Integrated Islets Distribution Program (IIDP) as described previously [7][8][9][10] . Human islets were cultured in RPMI 1640 medium with 10% FBS and 1% L-glutamine.…”

“…One such hormone is the adipocyte-derived hormone leptin, which regulates serum insulin levels by suppressing glucosestimulated insulin secretion (GSIS) from -cells [1][2][3][4][5] . Leptin does so by promoting trafficking of ATP-sensitive potassium (KATP) channels to the -cell membrane [6][7][8][9][10] . This increased surface expression of KATP channels increases the total KATP channel conductance thereby causing membrane hyperpolarization and reducing -cell electrical activity.…”

“…Leptin stimulates Src kinase to phosphorylate and potentiate NMDA receptor (NMDAR) activity, resulting in enhanced Ca 2+ influx that activates calcium/calmodulindependent kinase kinase  (CaMKK, which then phosphorylates and activates AMP-activated protein kinase (AMPK). Downstream of AMPK actin depolymerization occurs and results in increased trafficking of KATP channels to the plasma membrane [7][8][9][10] . We have shown previously that actin remodeling and subsequent KATP channel trafficking following AMPK activation requires protein kinase A (PKA).…”