Hyperinsulinemia is a risk factor for late-onset Alzheimer's disease (AD).In vitroexperiments describe potential connections between insulin, insulin signaling, and amyloid-β (Aβ), butin vivoexperiments are needed to validate these relationships under physiological conditions. First, we performed hyperinsulinemic-euglycemic clamps with concurrent hippocampal microdialysis in young, awake, behavingAPP_swe/PS1_dE9transgenic mice. Both a postprandial and supraphysiological insulin clamp significantly increased interstitial fluid (ISF) and plasma Aβ compared with controls. We could detect no increase in brain, ISF, or CSF insulin or brain insulin signaling in response to peripheral hyperinsulinemia, despite detecting increased signaling in the muscle. Next, we delivered insulin directly into the hippocampus of youngAPP/PS1mice via reverse microdialysis. Brain tissue insulin and insulin signaling was dose-dependently increased, but ISF Aβ was unchanged by central insulin administration. Finally, to determine whether peripheral and central high insulin has differential effects in the presence of significant amyloid pathology, we repeated these experiments in olderAPP/PS1mice with significant amyloid plaque burden. Postprandial insulin clamps increased ISF and plasma Aβ, whereas direct delivery of insulin to the hippocampus significantly increased tissue insulin and insulin signaling, with no effect on Aβ in old mice. These results suggest that the brain is still responsive to insulin in the presence of amyloid pathology but increased insulin signaling does not acutely modulate Aβin vivobefore or after the onset of amyloid pathology. Peripheral hyperinsulinemia modestly increases ISF and plasma Aβ in young and old mice, independent of neuronal insulin signaling.SIGNIFICANCE STATEMENTThe transportation of insulin from blood to brain is a saturable process relevant to understanding the link between hyperinsulinemia and AD.In vitroexperiments have found direct connections between high insulin and extracellular Aβ, but these mechanisms presume that peripheral high insulin elevates brain insulin significantly. We found that physiological hyperinsulinemia in awake, behaving mice does not increase CNS insulin to an appreciable level yet modestly increases extracellular Aβ. We also found that the brain of agedAPP/PS1mice was not insulin resistant, contrary to the current state of the literature. These results further elucidate the relationship between insulin, the brain, and AD and its conflicting roles as both a risk factor and potential treatment.