leptin and 2-aminoethoxydiphenyl-borate

Evidence suggests that the role played by the adipocyte-derived hormone leptin in female reproductive physiology is mediated in part by neurons located within the ventral premammillary nucleus (PMV). Leptin activates PMV neurons; however, the intracellular signaling pathway and channel(s) involved remain undefined. Notably, leptin's excitatory and inhibitory effects within hypothalamic and brainstem nuclei share the intracellular signaling cascade phosphoinositide 3 kinase (PI3K). Therefore, we assessed whether PI3K signaling is required for the acute effect of leptin to alter cellular activity of PMV neurons that express leptin receptors (LepR PMV neurons). Leptin caused a rapid depolarization in the majority of LepR PMV neurons in patch-clamp recordings of hypothalamic slices, while a subset of LepR PMV neurons were hyperpolarized in response to leptin. Data were obtained from both male and female mice and results demonstrate that the acute effect of leptin on LepR PMV neurons was identical for both sexes. Pharmacological inhibition of PI3K prevented the acute leptin-induced change in neuronal activity of LepR PMV neurons, indicating a PI3K-dependent mechanism of leptin action. Similarly, mice with genetically disrupted PI3K signaling in LepR PMV neurons failed to alter cellular activity in response to leptin. Moreover, the leptin-induced depolarization was dependent on a putative TRPC channel. In contrast, the leptin-induced-hyperpolarization required the activation of a putative Katp channel. Collectively, these results suggest that PI3K signaling in LepR PMV neurons is essential for leptin-induced alteration in cellular activity, and these data may suggest a cellular correlate in which leptin contributes to the initiation of reproductive development.

Topics: Androstadienes; Animals; Boron Compounds; Chromones; Female; Genes, Reporter; Hypothalamus; Imidazoles; KATP Channels; Leptin; Male; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Transgenic; Morpholines; Neurons; Patch-Clamp Techniques; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Subunits; Signal Transduction; TRPC Cation Channels; Wortmannin

Leptin can exert its potent appetite-suppressing effects via activation of hypothalamic proopiomelanocortin (POMC) neurons. It depolarizes POMC neurons via activation of a yet unidentified nonselective cation current. Therefore, we sought to identify the conductance activated by leptin using whole-cell recording in EGFP-POMC neurons from transgenic mice. The TRPC channel blockers SKF96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride), flufenamic acid, and 2-APB (2-aminoethyl diphenylborinate) potently inhibited the leptin-induced current. Also, lanthanum (La(3+)) and intracellular Ca(2+) potentiated the effects of leptin. Moreover, the diacylglycerol-permeable analog OAG (2-acetyl-1-oleoyl-sn-glycerol) failed to activate any TRPC current. Using a Cs(+)-gluconate-based internal solution, the leptin-activated current reversed near -20 mV. After replacement of external Na(+) and K(+) with Cs(+), the reversal shifted to near 0 mV, and the I/V curve exhibited a negative slope conductance at voltages more negative than -40 mV. Based on scRT-PCR, TRPC1 and TRPC4-7 mRNA were expressed in POMC neurons, with TRPC5 being the most prevalent. The leptin-induced current was blocked by the Jak2 inhibitor AG490, the PI3 kinase inhibitor wortmannin, and the phospholipase C inhibitors, U73122 and ET-18-OCH3. Notably, we identified PLCgamma1 transcripts in the majority of POMC neurons. Therefore, leptin through a Jak2-PI3 kinase-PLCgamma pathway activates TRPC channels, and TRPC1, 4, and 5 appear to be the key channels mediating the depolarizing effects of leptin in POMC neurons.

Topics: Animals; Appetite; Boron Compounds; Calcium Channel Blockers; Calcium Signaling; Female; Flufenamic Acid; Green Fluorescent Proteins; Hypothalamus; Imidazoles; Ion Channel Gating; Janus Kinase 2; Lanthanum; Leptin; Male; Membrane Potentials; Mice; Mice, Transgenic; Neurons; Organ Culture Techniques; Patch-Clamp Techniques; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phospholipase C gamma; Pro-Opiomelanocortin; Signal Transduction; Staining and Labeling; TRPC Cation Channels