Chase Garcia, Alexey Tomilov, Jose Sandoval, Gino Cortopassi
Abstract
Apolipoprotein E4 (ApoE4) is the strongest genetic risk factor for Alzheimer’s Disease (AD). ApoE4 confers defects in cerebral glucose metabolism years before cognitive impairments that may be relevant to health1,2. Recently it was demonstrated that ApoE4 binds the insulin receptor (IR) and impairs its signaling3.. Here we demonstrate that ApoE proteins bind the IR (figure 4), ApoE4 reduces downstream maximal insulin-stimulated phospho-Akt response (figure 2), and decreases further downstream mitochondrial glucose oxidation in N2a cells (figure 3). In conjunction, these data support the hypothesis that ApoE4 binds the insulin receptor, dysregulates p-Akt signaling, and this leads to downstream cerebral glucose impairment.
Methods:
- Constructed N2a neuroblastoma cells with stably transfected ApoE constructs (created by Dr. Alexey Tomilov, see poster “ApoE4 inhibits mitochondrial lipid oxidation in neural cells”)
- Dosed cells with varying concentrations of insulin and measured p-Akt response via quantitative western blotting capillary electrophoresis Jesstern machine
- Measured IR and ApoE binding by attaching IR via a His tag to sensor of Forte Octet biolayer interferometry machine (laser-based deflection assay). Measured binding of various concentrations of ApoE protein to IR.
Conclusions:
- ApoE3 increases p-Akt signaling the most, followed by ApoE4, ApoE2, GFP
- ApoE4 relative to ApoE3 has a decrease in p-Akt signaling
- ApoE affects downstream glucose metabolism (see poster by Jose Sandoval “ApoE allelic status affects both glycolytic rate and mitochondrial glucose oxidation in N2a cells” for full explanation)
- ApoE proteins directly bind to the insulin receptor