Insulin Confers Differing Effects on Neurite Outgrowth in Separate Populations of Cultured Dorsal Root Ganglion Neurons: The Role of the Insulin Receptor

Abstract

Apart from its pivotal role in the regulation of carbohydrate metabolism, insulin exerts important neurotrophic and neuromodulator effects on dorsal root ganglion (DRG) neurons. The neurite outgrowth-promoting effect is one of the salient features of insulin's action on cultured DRG neurons. Although it has been established that a significant population of DRG neurons express the insulin receptor (InsR), the significance of InsR expression and the chemical phenotype of DRG neurons in relation to the neurite outgrowth-promoting effect of insulin has not been studied. Therefore, in this study by using immunohistochemical and quantitative stereological methods we evaluated the effect of insulin on neurite outgrowth of DRG neurons of different chemical phenotypes which express or lack the InsR. Insulin, at a concentration of 10 nM, significantly increased total neurite length, the length of the longest neurite and the number of branch points of cultured DRG neurons as compared to neurons cultured in control medium or in the presence of 1 μM insulin. In both the control and the insulin exposed cultures, ∼43% of neurons displayed InsR-immunoreactivity. The proportions of transient receptor potential vanilloid type 1 receptor (TRPV1)-immunoreactive (IR), calcitonin gene-related peptide (CGRP)-IR and Bandeiraea simplicifolia isolectin B4 (IB4)-binding neurons amounted to ∼61%, ∼57%, and ∼31% of DRG neurons IR for the InsR. Of the IB4-positive population only neurons expressing the InsR were responsive to insulin. In contrast, TRPV1-IR nociceptive and CGRP-IR peptidergic neurons showed increased tendency for neurite outgrowth which was further enhanced by insulin. However, the responsiveness of DRG neurons expressing the InsR was superior to populations of DRG neurons which lack this receptor. The findings also revealed that besides the expression of the InsR, inherent properties of peptidergic, but not non-peptidergic nociceptive neurons may also significantly contribute to the mechanisms of neurite outgrowth of DRG neurons. These observations suggest distinct regenerative propensity for differing populations of DRG neurons which is significantly affected through insulin receptor signaling.

Keywords: calcitonin gene-related peptide; dorsal root ganglion neurons; insulin; insulin receptor; isolectin B4; neurite outgrowth; transient receptor potential vanilloid type 1 receptor.

FIGURE 1

The effects of insulin on neurite outgrowth of cultured adult rat dorsal root ganglion (DRG) neurons. (A–F) Fluorescence photomicrographs of β3-tubulin-stained DRG neurons illustrating the effects of insulin applied at concentrations of 10 nM (B) and 1 μM (C). The scale bar in (C) indicates 300 μm and applies to photomicrographs (A–C), the scale bar in (F) indicates 100 μm and applies to photomicrographs (D–F). (G–I) Quantitative morphometric evaluation of the effects of insulin on neurite outgrowth. Changes in total neurite length (G), maximum neurite length (H), and number of branch points (I) are shown as fold increase of the control. Values are expressed as mean ± standard error of the mean (SEM). ^∗Statistically significantly different from the control (p < 0.05). ^#Parameters of DRG neurons treated with 10 nM and 1 μM insulin, respectively, are significantly different (p < 0.05).

FIGURE 2

Neurochemical phenotypes of cultured adult rat dorsal root ganglion (DRG) neurons examined. (A) Large pie chart shows the percentage distribution of populations of DRG neurons showing immunoreactivities for the insulin receptor (InsR) and the transient receptor potential vanilloid type 1 receptor (TRPV1). Small pie charts show the relative proportions of TRPV1-immunoreactive (IR) neurons which display colocalization with the InsR and, conversely, the relative proportions of InsR-IR DRG neurons which display colocalization with TRPV1. (B) Large pie chart shows the percentage distribution of populations of DRG neurons showing immunoreactivities for the InsR and calcitonin gene-related peptide (CGRP). Small pie charts show the relative proportions of CGRP-IR DRG neurons which display colocalization with the InsR and, conversely, the relative proportions of InsR-IR DRG neurons which display colocalization with CGRP. (C) Large pie chart shows the percentage distribution of populations of DRG neurons showing immunoreactivity for the InsR and binding of the isolectin B4 (IB4). Small pie charts show the relative proportions of IB4-binding neurons which display colocalization with the InsR and, conversely, the relative proportions of InsR-IR DRG neurons which display colocalization with IB4.

FIGURE 3

Size-frequency distribution histogram of the total and the insulin receptor (InsR)-immunopositive and -negative populations of adult rat cultured dorsal root ganglion (DRG) neurons. Size-frequency distribution histogram shows the cross-sectional areas of the total DRG neuronal population (white bars), the InsR-immunopositive (black bars) and the InsR-immunonegative (gray bars) populations in the control cultures.

FIGURE 4

The effects of insulin (10 nM, 48 h) on neurite outgrowth of insulin receptor (InsR)-positive and InsR-negative cultured adult rat dorsal root ganglion (DRG) neurons. (A–C) Fluorescence photomicrographs of cultured adult rat dorsal root ganglion (DRG) neurons double-stained for β3-tubulin- and InsR-immunoreactivities. The scale bar in (C) indicates 100 μm and applies to all photomicrographs. (D–F) Quantitative morphometric evaluation of the effects of insulin on neurite outgrowth of InsR-positive and InsR-negative populations of DRG neurons. Changes in total neurite length (D), maximum neurite length (E) and number of branch points (F) are shown. Values are expressed as mean ± standard error of the mean (SEM). ^∗Statistically significantly different from the control (p < 0.05). ^#Parameters of InsR-positive and InsR-negative DRG neurons are significantly different (p < 0.05).

FIGURE 5

The effects of insulin (10 nM, 48 h) on neurite outgrowth of cultured adult rat dorsal root ganglion (DRG) neurons. (A–D) Fluorescence photomicrographs of cultured adult rat dorsal root ganglion (DRG) neurons triple-stained for β3-tubulin, insulin receptor (InsR) and transient receptor potential vanilloid type 1 receptor (TRPV1). The scale bar in (D) indicates 100 μm and applies to all photomicrographs. (E–G) Quantitative morphometric evaluation of the effects of insulin on neurite outgrowth of InsR-positive and InsR-negative populations of TRPV1-positive and TRPV1-negative DRG neurons. Changes in total neurite length (E), maximum neurite length (F), and number of branch points (G) are shown. Values are expressed as mean ± standard error of the mean (SEM). ^∗, ^#, and ^x: Statistically significantly different from the corresponding control and/or insulin-treated DRG neuron populations (p < 0.05).

FIGURE 6

The effects of insulin (10 nM, 48 h) on neurite outgrowth of cultured adult rat dorsal root ganglion (DRG) neurons. (A–D) Fluorescence photomicrographs of cultured adult rat dorsal root ganglion (DRG) neurons triple-stained for β3-tubulin, insulin receptor (InsR) and calcitonin gene-related peptide (CGRP). The scale bar in (D) indicates 100 μm and applies to all photomicrographs. (E–G) Quantitative morphometric evaluation of the effects of insulin on neurite outgrowth of InsR-positive and InsR-negative populations of CGRP-positive and CGRP-negative DRG neurons. Changes in total neurite length (E), maximum neurite length (F) and number of branch points (G) are shown. Values are expressed as mean ± standard error of the mean (SEM). ^∗, ^#, and ^x: Statistically significantly different from the corresponding control and/or insulin-treated DRG neuron populations (p < 0.05).

FIGURE 7

The effects of insulin (10 nM, 48 h) on neurite outgrowth of cultured adult rat dorsal root ganglion (DRG) neurons. (A–D) Fluorescence photomicrographs of cultured adult rat dorsal root ganglion (DRG) neurons triple-stained for β3-tubulin, insulin receptor (InsR) and isolectin B4 (IB4). The scale bar in (D) indicates 100 μm and applies to all photomicrographs. (E–G) Quantitative morphometric evaluation of the effects of insulin on neurite outgrowth of InsR-positive and InsR-negative populations of IB4-positive and IB4-negative DRG neurons. Changes in total neurite length (E), maximum neurite length (F) and number of branch points (G) are shown. Values are expressed as mean ± standard error of the mean (SEM). ^∗, ^#, and ^x: Statistically significantly different from the corresponding control and/or insulin-treated DRG neuron populations (p < 0.05).