Insulin-degrading enzyme as a downstream target of insulin receptor signaling cascade: implications for Alzheimer's disease intervention

Abstract

Insulin-degrading enzyme (IDE) is one of the proteins that has been demonstrated to play a key role in degrading beta-amyloid (Abeta) monomer in vitro and in vivo, raising the possibility of upregulating IDE as an approach to reduce Abeta. Little is known, however, about the cellular and molecular regulation of IDE protein. Because one of the main functions of IDE is to degrade insulin, we hypothesized that there is a negative feedback mechanism whereby stimulation of insulin receptor-mediated signaling upregulates IDE to prevent chronic activation of the pathway. We show that treatment of primary hippocampal neurons with insulin increased IDE protein levels by approximately 25%. Insulin treatment also led to phosphatidylinositol-3 (PI3) kinase activation evidenced by Akt phosphorylation, which was blocked by PI3 kinase inhibitors, wortmannin and LY 294002. Inhibition of PI3 kinase abolished the IDE upregulation by insulin, indicating a cause-effect relationship between insulin signaling and IDE upregulation. Further support for this link was provided by the findings that deficient insulin signaling (decreased PI3 kinase subunit P85) was correlated with reduced IDE in Alzheimer's disease (AD) brains and in Tg2576 Swedish amyloid precursor protein transgenic mice fed a safflower oil-enriched ("Bad") diet used to accelerate pathogenesis. Consistent with IDE function in the degradation of Abeta monomer, the IDE decrease in the Bad diet-fed Tg2576 mice was associated with increased Abeta monomer levels. These in vitro and in vivo analyses validate the use of enhanced CNS insulin signaling as a potential strategy for AD intervention to correct the IDE defects occurring in AD.

Figure 1.

Insulin induces IDE upregulation in primary hippocampal neurons through the PI3 kinase-Akt signaling pathway. Hippocampal neurons were treated with different doses of insulin for 24 hr, followed by determination of IDE by Western immunoblotting (A). Neurons were then treated with 20 nm insulin for 24 hr with or without PI3 kinase inhibitors wortmannin (Wort; 50 nm) or LY 294002 (LY; 25 μm), followed by determination of IDE and phospho-Akt levels by Western immunoblotting. LDH release was used as an index for cell viability (B). ^*p ≤ 0.05 relative to control.

Figure 2.

Immunocytochemical images showing insulin-induced IDE upregulation and PI3 kinase activation in vitro in primary hippocampal neurons. Hippocampal neurons were treated with 20 nm insulin with or without PI3 kinase inhibitors wortmannin (Wort; 50 nm) or LY 294002 (LY; 25 μm) for 24 hr, followed by determination of IDE and phospho-Akt levels by immunocytochemical staining. DAPI was used to stain neuronal nuclei. NIH Image software was used to quantify the fluorescence. Scale bar, 50 μm. ^*p ≤ 0.05 relative to control.

Figure 3.

Both IDE and p85 (a PI3 kinase subunit) were decreased in the hippocampus (A) and temporal cortex (B) of AD brains compared with normal brains. IDE and p85 levels were significantly correlated in both hippocampus and temporal cortex, providing an in vivo support for the causal relationship between PI3 kinase and IDE. ^*p ≤ 0.05 relative to normal. IDE levels in the hippocampus (C) and temporal cortex (D) were also plotted against the number of copies of ApoE4 in the human subjects.

Figure 4.

Both IDE and p85 mRNA and protein levels were decreased in Tg2576 transgenic mice raised with safflower oil-based (Bad) diet compared with mice on standard diet, and the mRNA and protein levels of IDE and p85 were significantly correlated (A). In the Bad (B) diet-fed mice, the Aβ monomer levels were significantly increased compared with animals raised on standard (S) diet, and the levels of Aβ monomer and IDE were significantly inversely correlated, supporting the significance of IDE in Aβ degradation (B). ^*p ≤ 0.05 relative to standard.