The Effects of Insulin on Immortalized Rat Schwann Cells, IFRS1

Abstract

Schwann cells play an important role in peripheral nerve function, and their dysfunction has been implicated in the pathogenesis of diabetic neuropathy and other demyelinating diseases. The physiological functions of insulin in Schwann cells remain unclear and therefore define the aim of this study. By using immortalized adult Fischer rat Schwann cells (IFRS1), we investigated the mechanism of the stimulating effects of insulin on the cell proliferation and expression of myelin proteins (myelin protein zero (MPZ) and myelin basic protein (MBP). The application of insulin to IFRS1 cells increased the proliferative activity and induced phosphorylation of Akt and ERK, but not P38-MAPK. The proliferative potential of insulin-stimulated IFRS1 was significantly suppressed by the addition of LY294002, a PI3 kinase inhibitor. The insulin-stimulated increase in MPZ expression was significantly suppressed by the addition of PD98059, a MEK inhibitor. Furthermore, insulin-increased MBP expression was significantly suppressed by the addition of LY294002. These findings suggest that both PI3-K/Akt and ERK/MEK pathways are involved in insulin-induced cell growth and upregulation of MPZ and MBP in IFRS1 Schwann cells.

Keywords: Akt; Schwann cells; extracellular-signal-regulated kinase (ERK); insulin; myelin associated glycoprotein; myelin basic protein; myelin protein zero.

Figure 1

(A) Differential interference contrast (DIC) microscopic image of IFRS1. (B) Confocal immunofluorescence analysis of IFRS1 treated with anti-IR-beta rabbit mAb labeled with Alexa Fluor 568-conjugated phalloidin (red). Blue pseudo color-DAPI. (C) IR-beta was identified by Western blot analyses using the anti-IR-beta antibody.

Figure 2

(A–D): The phosphorylation of Akt, MEK, extracellular signal-regulated kinase (ERK), and 38-mitogen-activated protein kinase (MAPK) by insulin. Insulin stimulation (100 nM) was conducted for the indicated time. (E–H): The concentration-dependent phosphorylation of Akt, MEK, ERK, and p38-MAPK by insulin.

Figure 3

Insulin promotes the proliferation of IFRS1. Cell growth was arrested using serum-free Dulbecco’s Modified Eagle Medium (DMEM) for 12 h. After pre-incubation with LY294002 (30–3000 nM) or PD98059 (30–3000 nM), cells were incubated with insulin (100 nM). (A) Cell proliferation was assessed using an MTT assay. (B) Cell proliferation was assessed by CCK-8. The results are shown as the mean ± standard error of the mean (SEM). * p < 0.01 versus the control. (C) Representative images of the IFRS1 observed under phase-contrast microscopy. (Bar = 200 μm).

Figure 4

Data on insulin-stimulated myelin protein zero (MPZ) expression through various inhibitors. (A) Western blot data showing the expression of MPZ through insulin stimulation, and the effect of LY294002 and PD98059 on MPZ. The results of one of three experiments with similar results are shown. * p < 0.05 versus insulin (DMSO control). (B) Immunofluorescence-stained image of MPZ. Green indicates MPZ and blue indicates DAPI (Bar = 100 μm).

Figure 5

Data on insulin-stimulated myelin basic protein (MBP) expression by various inhibitors. (A) Western blot data showing the expression of MBP by insulin stimulation and the effect of LY294002 and PD98059 on MBP. The results of one of three experiments with similar results are shown. * p < 0.05 versus insulin (DMSO control). (B) Immunofluorescence-stained image of MBP. Green indicates MBP and blue indicates DAPI (Bar = 100 μm).

Figure 6

Proposed scheme for insulin-stimulated signaling of the proliferation and the production of myelin-related proteins. Insulin promotes the MPZ expression of IFRS1 via MEK/ERK and MBP expression of PI3-K/Akt, whereas insulin promotes the proliferation of IFRS1 via PI3-K/Akt.