Leptin transiently antagonizes ghrelin and long-lastingly orexin in regulation of Ca2+ signaling in neuropeptide Y neurons of the arcuate nucleus

Figure 1 Ghrelin- and orexin-induced [Ca²⁺]_i increases were suppressed by leptin in a transient or long-lasting manner in NPY neurons. A-D: Ghrelin and orexin-A at 10^-10 mol/L increased [Ca²⁺]_i in single neurons isolated from the ARC, and these [Ca²⁺]_i increases were suppressed by administration of leptin at 10^-12 mol/L (A, C) and 10^-14 mol/L (D). Following [Ca²⁺]_i measurements, the neurons in (A) and (C) were proved to contain NPY by immunocytochemistry using anti-NPY antibody (A, C; right panels). The bars indicate 10 μm. E: Leptin inhibited ghrelin-induced [Ca²⁺]_i increases in a greater number of neurons in 2-7 min after administration than in 10-15 min. In contrast, leptin inhibited orexin-A-induced [Ca²⁺]_i increases in the majority of neurons in 2-7 min and 10-15 min after administration. The numbers above the bars indicate the number of neurons inhibited by leptin over that responded to ghrelin or orexin-A. F: The number of neurons whose [Ca²⁺]_i responses to ghrelin or orexin-A were suppressed by leptin in either a transient or long-lasting manner is expressed by percentage. The numbers above the points indicate the number of neurons inhibited by leptin in the specified manner over that responded to ghrelin or orexin-A.

Figure 2 Leptin suppresses ghrelin-induced and orexin-induced [Ca²⁺]_i increases via different signaling pathways in NPY neurons. A-G: Effects of inhibitors for signaling molecules on the leptin action against ghrelin and orexin. Preincubation for 1 h with 50 μmol/L LY294002, an inhibitor for PI3-kinase, interfered with leptin action to suppress [Ca²⁺]_i responses to ghrelin (A), but not orexin (B). Preincubation for 1 h with 100 nmol/L wortmannin, another PI3-kinase inhibitor, little affected the leptin suppression of orexin-induced [Ca²⁺]_i increases (C). Preincubation for 30 min with 10 μmol/L milrinone, a PDE3 inhibitor, blocked the leptin action to suppress [Ca²⁺]_i responses to ghrelin (D), but not orexin (E). Preincubation for 30 min with 10 μmol/L U0126, an inhibitor for MAP-kinase, little affected the leptin action to suppress orexin-induced [Ca²⁺]_i increases (F). Preincubation for 30 min with a STAT3 inhibitor peptide at 500 μmol/L, did not significantly alter the leptin suppression of orexin-induced [Ca²⁺]_i increases (G). H: The number of neurons whose [Ca²⁺]_i responses to ghrelin or orexin were suppressed by leptin in the presence of inhibitors is expressed by percentage. The numbers above the bars indicate the number of neurons inhibited by leptin over that responded to ghrelin or orexin-A. I: Reduction of amplitudes of [Ca²⁺]_i responses to ghrelin or orexin-A by leptin is expressed by percentage. ^aP < 0.05, ^bP < 0.01 vs control. The data regarding ghrelin in H and I were in part taken from our previous paper[17].

Figure 3 Prior treatment with leptin suppressed [Ca²⁺]_i responses to subsequent administration of orexin, but not ghrelin, in NPY neurons. A, C: Repeated administrations of ghrelin or orexin-A at 10^-10 mol/L increased [Ca²⁺]_i twice in single ARC neurons; B, D: Prior administration of leptin failed to inhibit the effect of ghrelin added subsequently (B), but inhibited the effect of orexin-A (D); E: The number of neurons in which [Ca²⁺]_i responses to the second ghrelin or orexin addition were suppressed by prior administration of leptin or HKRB (Control) is expressed by percentage. The numbers above the bars indicate the number of neurons whose second responses were inhibited over that exhibited first responses to ghrelin or orexin-A.