IRs and IRS2 are expressed in NG neurons, and inhibition of IRS2 and PI3K but not MAPK suppresses insulin-induced [Ca²⁺]_i increases in NG neurons.

<p><b><i>A</i></b>: Neurons immunoreactive to IR-β, revealed by Alexa 488 fluorescence, were observed in the section of mouse NG. The picture is representative of 13 sections from 4 mice. The right picture is the magnification of the area marked with dotted line in the left picture. <b><i>B</i></b>: mRNA expressions for IRs and IRS2 in NGs. Representative electrophoretic patterns of RT-PCR products of IR (left) and IRS2 (right) mRNAs in NGs. M = size maker, (−) = RT (−) as a negative control, (+) = RT (+). <b><i>C∼E</i></b>: Effects of insulin (10⁻⁷ M), CCK (10⁻⁸ M), and CAP (10⁻⁷ M) on [Ca²⁺]_i in NG neurons derived from wild-type C57BL/6J (<i>C</i>) and IRS2 knockout mice (<i>D</i>). The [Ca²⁺]_i response to insulin, but not CCK-8 and CAP, were markedly attenuated in the NG neurons of IRS2 knockout mice (<i>D</i>, n = 9) as compared with wild type (<i>C</i>, n = 14). The amplitude of [Ca²⁺]_i response to each regent is normalized to that to KCl in each neuron and expressed as relative values (percentage) (<i>E</i>). The numbers on each bar indicate the numbers of neurons that responded to insulin, CCK-8 or CAP. The amplitude of [Ca²⁺]_i responses to insulin were suppressed by IRS2-deficiency. <b><i>F∼H</i></b>: Effects of a PI3K inhibitor LY294002 at 50 µM (LY, pretreatment for 1 hr, <i>F</i>) and a MAPK inhibitor U0126 at 10 µM (U, pretreatment for 0.5 hr, <i>G</i>) on insulin-induced [Ca²⁺]_i increases in NG neurons. The [Ca²⁺]_i traces of control experiments (C) are shown in Fig. 1E. In these three conditions, the amplitude of [Ca²⁺]_i response to each regent is normalized to that to KCl in each neuron and expressed as relative values (percentage) (<i>H</i>). The numbers on each bar indicate the numbers of neurons that responded insulin, CCK-8 or CAP. [Ca²⁺]_i responses to insulin were suppressed by LY but not U. *<i>P</i><0.05, **<i>P</i><0.01 by paired t-test (<i>E</i>) and one-way ANOVA followed by Dunnett’s test vs. control (<i>H</i>).