| Abstract No.: | C-E3161 |
| Country: | Canada |
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| Title: | EXPRESSION, DISTRIBUTION, AND ACTIVITY OF THE INSULIN RECEPTOR IN CULTURED HIPPOCAMPAL SLICES
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| Authors/Affiliations: | 1 John Mielke*; 1 Geoffrey Mealing;
1 National Research Council of Canada, Institute for Biological Sciences, Ottawa, ON, Canada
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| Content: | Objectives: The insulin receptor (IR) is distributed in a region-specific fashion throughout the brain, and plays diverse roles that are distinct from its participation in glucose regulation. Behavioural experiments in both humans and animals have shown that insulin can improve cognition, whilst impairment of central insulin signaling has been associated with Alzheimer’s disease. To further understanding of the cellular processes linking the IR with learning and memory, the protein expression, cellular distribution, and ligand-binding characteristics of the receptor were examined in organotypic hippocampal slice cultures (OHSCs).
Materials & Methods: OHSCs were prepared by isolating hippocampi from P8 Sprague-Dawley rat pups, cutting 400 μm sections with a McIIwain tissue chopper, and maintaining the slices on semiporous inserts within a humidified 5% CO2 incubator1. At one week intervals, after insulin stimulation, or following application of the membrane-impermeable cross-linking reagent BS3 (applied for 15 min at 4oC), OHSCs were homogenised with non-ionising lysis buffer supplemented with protease inhibitors. After SDS-PAGE, proteins were transferred to PVDF membranes, and immunoblots probed with antibodies of interest following standard techniques. For the insulin binding studies, insulin conjugated to a fluorophore (INS-FITC) was applied to OHSCs, which were then homogenised and assessed fluorometrically2.
Results: The IR is a heterotetramer formed from two alpha and two beta subunits. Over four weeks in culture, levels of the transmembrane beta subunit protein were shown to decline by approximately 60%. As a result, subsequent experiments were completed with slices after only a week in culture. Brief insulin stimulation of OHSCs resulted in a concentration-dependent increase in serine phosphorylation of Akt/PKB, a principal metabolic effector, indicating fidelity of signal transduction. A general assessment of cellular distribution by cross-linking of surface proteins revealed the IR to have a high degree of surface expression, albeit less so than certain ionotropic receptors. To study neural insulin receptor binding activity, INS-FITC (100 nM) was applied to OHSCs that had been pretreated with unlabelled insulin at either various concentrations or for various lengths of time. Pre-application of unlabelled insulin (100, 500, or 5000 nM for 1 h) increased the amount of INS-FITC binding in a concentration-dependent fashion, but pretreatment with 5000 nM unlabelled insulin for longer periods (2 and 3 h) was not able to further increase the amount of INS-FITC labeling.
Conclusions: The first part of the study described a number of basic features of the IR within OHSCs, and confirmed the utility of the in vitro model for understanding how the receptor contributes to hippocampal function. The second part of the study found that the binding affinity for INS-FITC increased with receptor occupancy. Consequently, neural IRs, unlike their peripheral counterparts, display a hormone-receptor interaction that suggests the presence of a positive cooperative effect.
1Mielke et al. (2005) Dev. Brain Res. 160 275-286.
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