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 that, when phosphorylated by Src family kinases, potentiate NMDAR activity. We found that leptin increases phosphorylation of Tyr-418 in Src, an indicator of kinase activation. Pharmacological inhibition of Src or overexpression of a kinase-dead Src mutant prevented the effect of leptin, whereas a Src kinase activator peptide mimicked it. Using mutant GluN2A overexpression, we show that Tyr-1292 and Tyr-1387 but not Tyr-1325 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.
Highlights
Leptin is an adipocyte-produced hormone that plays a key role in body weight regulation
Multiple lines of evidence support our conclusion that leptin modulation of b-cell membrane potential requires the phosphorylation of GluN2Acontaining NMDA subtype glutamate receptors (NMDARs) by Src kinase
Leptin increased phosphorylation of Src at Tyr-418, a site that is required for its catalytic activity [45, 46], and dominantnegative suppression of Src activity via a kinase-dead mutant prevented the ability of leptin to increase surface KATP channels
Summary
In rodent pancreatic b-cell lines as well as primary human b-cells, leptin induces membrane hyperpolarization at glucose concentrations that depolarize b-cell membrane potential [3, 7,8,9, 19]. Application of 50 mM NMDA in the bath solution to directly activate NMDARs in b-cells from the same donors used to test leptin response did induce significant hyperpolarization (DVm = 222.0 6 8.6 mV, n = 9) (Fig. 8, C and D), indicating that NMDAR function and downstream signaling events were not compromised as was observed in b-cells from db/db mice. These results show that b-cells from a sample of obese diabetic donors were leptin-resistant but retained the ability to hyperpolarize in response to direct NMDAR activation
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