Hypothalamic actions and interactions of alcohol and IGF-1 on the expression of glial receptor protein tyrosine phosphatase-β during female pubertal development

Abstract

Background: Hypothalamic glial-neuronal communications are important for the activation of luteinizing hormone releasing hormone (LHRH) secretion at the time of puberty. As we have shown that alcohol (ALC) diminishes prepubertal LHRH secretion and delays puberty, we first assessed the effects of short-term ALC administration on the basal expression of a specific gene family involved in glial-neuronal communications. Second, as insulin-like growth factor-1 (IGF-1) is a critical regulator of LHRH secretion and the pubertal process, we then assessed whether IGF-1 could induce the expression of these signaling genes and determine whether ALC can block this affect.

Methods: Immature female rats were fed a liquid diet containing ALC for 6 days beginning when 27 days old. Control animals received either the companion isocaloric liquid diet or rat chow and water. Animals were decapitated on day 33, in the late juvenile stage of development. Medial basal hypothalamic (MBH) tissues were obtained for gene and protein analyses of glial receptor protein tyrosine phosphatase-β (RPTPβ) and the 2 neuronal components, contactin and contactin-associated protein 1 (Caspr1). In the second experiment, IGF-1 was administered into the third ventricle (3V) and the MBH removed 6 hours after peptide delivery, and the above-mentioned 3 genes were analyzed by real-time PCR. To determine whether this action was affected by ALC, immature female rats were administered either ALC (3 g/kg) or water via gastric gavage at 0900 hours. At 1030 hours, the ALC and control groups were subdivided such that half of the animals were injected into the 3V with IGF-1 and the other half with an equal volume of saline. Rats were killed 6 hours after the IGF-1 injection and MBHs collected.

Results: Real-time PCR showed that when compared with control animals, ALC caused a marked decrease (p < 0.001) in the basal expression of the RPTPβ gene, but did not affect the expression of either contactin or Caspr1. Likewise, analysis by Western blotting demonstrated that ALC caused suppressed (p < 0.001) levels of the RPTPβ protein, with the expressions of both contactin and Caspr1 proteins being unaltered. In the second experiment, results showed that only the RPTPβ gene was stimulated (p < 0.05) by IGF-1 in the MBH 6 hours after peptide delivery. Assessments revealed that the IGF-1 induced increase (p < 0.01) in the expression of the RPTPβ gene was blocked by the presence of ALC.

Conclusions: Prepubertal ALC exposure is capable of interfering with hypothalamic glial-neuronal communications by suppressing the synthesis of the glial product, RPTPβ, which is required for binding to the contactin-Caspr1 complex on LHRH neuronal terminals, thus suggesting that this action of ALC contributes to its detrimental effects on the pubertal process.

Figure 1

Effect of short-term ALC exposure on basal RPTPβ (A), contactin (B) and Caspr1 (C) gene expressions in the MBH of prepubertal female rats as determined by real-time PCR. Note that ALC caused a marked decrease in basal RPTPβ gene expression compared with control animals, but did not alter the gene expression of either contactin or Caspr1. The respective bars illustrate the mean (±SEM) of an N of 12–13 per group. ***p<0.001 versus control.

Figure 2

Effect of short-term ALC exposure on basal RPTPβ, contactin and Caspr1 protein expressions in the MBH of prepubertal rats. (A) Representative Western immunoblot of RPTPβ and β-actin proteins in the MBH isolated from control (lanes 1–3) and ALC-treated (lanes 4–6) animals. (B) Densitometric quantitation of all the bands from 2 blots assessing RPTPβ protein in the MBH. (C) Representative Western immunoblot of contactin and β-actin proteins in the MBH isolated from control (lanes 1–3) and ALC-treated (lanes 4–6) animals. (D) Densitometric quantitation of all the bands from 2 blots assessing contactin protein in the MBH. (E) Representative Western immunoblot of Caspr1 and β-actin proteins in the MBH isolated from control (lanes 1–3) and ALC-treated (lanes 4–6) animals. (F) Densitometric quantitation of all the bands from 2 blots assessing Caspr1 protein in the MBH. Note that ALC caused a marked decrease in RPTPβ protein expression compared with control animals, but did not alter the protein expression of either contactin or Caspr1.The respective bars illustrate the mean (±SEM) of an N of 10 for control and an N of 6 for ALC. All of the above data were normalized to the internal control β-actin protein. ***p<0.001 versus control.

Figure 3

Effect of IGF-1 on basal RPTPβ (A), contactin (B), and Caspr1 (C) gene expressions in the MBH of prepubertal female rats as determined by real-time PCR. IGF-1 (200 ng) was injected into the third ventricle and gene expressions in the MBH were assessed at 6h post-injection. Note that IGF-1 induced a significant increase in RPTPβ gene expression compared with control animals, but did not affect the expression of either contactin or Caspr1.The respective bars illustrate the mean (±SEM) of an N of 7 per group for RPTPβ and an N of 8–10 per group for contactin and Caspr1. *p<0.05 versus control.

Figure 4

Effect of acute ALC exposure on basal and IGF-1 stimulated RPTPβ gene expressions in the MBH as determined by real-time PCR. The RPTPβ gene expression was assessed in the MBH at 6 h post IGF-1 injection. The IGF-1 induced an increase in the expression of the RPTPβ gene compared with the basal level shown in the controls. Note that the basal expression of the RPTPβ gene was not altered by ALC alone, but that the presence of ALC blocked the IGF-1 induced expression of the gene. The respective bars illustrate the mean (±SEM) of an N of 8–10 per group. **p<0.01 as assessed by ANOVA, with post-hoc testing using Student-Newman-Keuls.

Figure 5

Schematic drawing showing glia-LHRH neuronal association and sites of ALC effects on RPTPβ in the median eminence of juvenile female rats. For clarity, details of other downstream pathways in this region are not shown. IGF-1, insulin-like growth factor-1; EGF, epidermal growth factor; ALC, alcohol, IGFR, Insulin-like growth factor receptor; PGE₂, prostaglandin-E₂; LHRH, luteinizing hormone releasing hormone; RPTPβ, receptor protein tyrosine phosphatase β; Caspr1, contactin associated protein-1; ER, estrogen receptor; TGF, transforming growth factor; BBB, blood brain barrier