Sustained Impairments in Brain Insulin/IGF Signaling in Adolescent Rats Subjected to Binge Alcohol Exposures during Development

Abstract

Background: Chronic or binge ethanol exposures during development can cause fetal alcohol spectrum disorder (FASD) which consists of an array of neurobehavioral deficits, together with structural, molecular, biochemical, and neurotransmitter abnormalities in the brain. Previous studies showed that perinatal neurodevelopmental defects in FASD are associated with inhibition of brain insulin and insulin-like growth factor (IGF) signaling. However, it is not known whether sustained abnormalities in adolescent brain structure and function are mediated by the same phenomena.

Aims: Using an early postnatal (3^rd trimester equivalent) binge ethanol exposure model, we assessed neurobehavioral function, structure, and the integrity of insulin/IGF signaling in young adolescent cerebella.

Methods: Long Evans male rats were treated with 50 µl of saline (vehicle) or 2 mg/kg of ethanol by i.p. injection on postnatal days (P) 2, 4, 6, and 8. On P19-20, rats were subjected to rotarod testing of motor function, and on P30, they were sacrificed to harvest cerebella for histological, molecular, and biochemical studies.

Results: Binge ethanol exposures impaired motor function, caused sustained cerebellar hypocellularity, and reduced neuronal and oligodendrocyte gene expression. These effects were associated with significant deficits in insulin and IGF signaling, including impaired receptor binding, reduced Akt, and increased GSK-3β activation.

Conclusions: FASD-associated neurobehavioral, structural, and functional abnormalities in young adolescent brains may be mediated by sustained inhibition of insulin/IGF-1 signaling needed for cell survival, neuronal plasticity, and myelin maintenance.

Keywords: Adolescence; Brain development; Brain insulin resistance; Central nervous system; Cerebellum; Fetal alcohol syndrome; Insulin signaling; Motor function; Receptor binding.

Figure 1

Long-term effects of early postnatal binge ethanol exposure on rotarod motor performance. Long Evans rat pups were treated with 50 µl i.p. injections of saline (vehicle) or 2.0 mg/kg ethanol in saline on postnatal days 2, 4, 6, and 8. On postnatal 19, rats (N=12/group) were subjected to 10 rotarod test trials in which speed of the rotating rod was incremented with each trial. Latency to fall was recorded. Data from Trials 1–3 (2–5 rpm), Trials 4–6 (5–7 rpm), or Trials 7–10 (8–10 rpm) were culled and analyzed using the Mann-Whitney test. Significant inter-group differences are shown in the panels.

Figure 2

Sustained structural abnormalities in adolescent cerebella following early postnatal binge ethanol exposures. Long Evans rat pups were treated with 50 µl i.p. injections of saline (vehicle) or 2.0 mg/kg ethanol in saline on postnatal days 2, 4, 6, and 8. Rats were sacrificed on P30 and cerebella were fixed and embedded in paraffin. Histological sections were stained with Hematoxylin and Eosin. (A, B) Low (original, 40×) and (C, D) high (original, 160×) magnification images demonstrating (A, B) control cerebella with long slender folia with uniform thickness of the molecular layer (ml), and well-populated Purkinje (pc) and granule cell (gc) layers in cortex, compared with (C, D) ethanol-exposed cerebella, which had shallow, blunted and simplified folia with irregular thickness of the molecular layer, irregular thinning of the granule cell layer (gcl) and white matter (wm) cores, and numerous gaps corresponding with loss of neurons in the Purkinje cell layer. White lines span the thicknesses of the granule cell layers.

Figure 3

Early postnatal binge ethanol exposures alter neuronal and glial gene expressions in early adolescent cerebella. RNA extracted from cerebella (N=8 samples per group) was reverse transcribed, and the cDNAs were used to measure gene expression by qPCR analysis. Results were normalized to 18S rRNA measured in parallel reactions. Graphs depict relative levels of gene expression for (A) neuronal Hu, (B) myelin-associated glycoprotein-1 (MAG-1), (C) glial fibrillary acidic protein (GFAP), (D) allograft inflammatory factor -1 (AIF), (E) acetylcholinesterase (AChE), and (F) choline acetyltransferase (ChAT), and. Inter-group comparisons were made using Student t-tests. Significant P-values are shown over the graphs.

Figure 4

Effects of early postnatal binge ethanol exposures on expression of insulin, insulin-like growth factor-1 (IGF-1), IRS-2, and insulin receptor substrate genes in young adolescent cerebella. RNA extracted from cerebella (N=8 samples per group) was reverse transcribed, and the cDNAs were used to measure gene expression corresponding to the (A) insulin, IGF-1, and IGF-2 polypeptides, (B) insulin, IGF-1, and IGF-2 receptors, and (C) IRS1, IRS2, and IRS4. Results were normalized to 18S rRNA measured in parallel reactions. Graphs depict relative levels of gene expression Inter-group comparisons with respect to trophic factors, receptors, or IRS molecules were made by repeated measures two-way ANOVA tests with the Bonferroni post hoc significance test. Significant P-values are shown over the graphs.

Figure 5

Sustained insulin and IGF-1 resistance in young adolescent cerebella following early postnatal binge ethanol exposures Cerebella protein homogenates were used to measure immunoreactivity to (A) insulin receptor (IR), (D) IGF-1R, (G) IRS-1, (B) pYpY1162/1163-IR, (E) pYpY1135/1136-IGF-1R, (H) pS312-IRS-1 using the bead-based Akt and phospho-specific Akt pathway multiplex ELISA kits. Phospho-/total protein ratios for (C) IR, (F) IGF-1R, and (I) IRS-1 were calculated. Comparisons (N=8 samples per group) were made using Student T-tests. Significant differences are indicated within the panels.

Figure 6

Effects of early postnatal binge ethanol exposures on signaling through Akt and GSK-3β in young adolescent cerebella. Cerebellar protein homogenates were used to measure immunoreactivity to (A) Akt, (B) pS473-Akt, (D) GSK-3β, and (E) pS9-GSK-3β using bead based total Akt and phospho-specific Akt multiplex ELISA kits. In addition, relative degrees of phosphorylation represented by (C) pS473/Total Akt and (F) pS9/Total GSK-3β were calculated. Inter-group comparisons were made using Student T-tests. Significant differences are indicated within the panels.

Figure 7

Impaired insulin and IGF-1 receptor binding in young adolescent cerebella following early postnatal binge ethanol exposures Competitive saturation binding assays were performed by incubating cerebellar membrane protein extracts with 0.5–500 pM [125I]-labeled insulin, IGF-1, or IGF-2 as tracer, in the presence or absence of 100 nM cold ligand. Membrane bound tracer was precipitated and radioactivity in the supernatants (containing free ligand) and pellets (containing bound ligand) was measured in a gamma counter. Graphs depict specific binding (fmol/mg protein) ± S.D. relative to pM input of radiolabeled (A–B) insulin, (C,D) IGF-1, and (E,F) IGF-2 in (A,C,E) control (vehicle-treated) and (B,D,F) ethanol-exposed samples. The calculated binding indices and inter-group statistical comparisons are provided in Table 1.