Insulin fails to mitigate amyloid pathology but modulates intestinal morphology in the 5xFAD model revealed by synchrotron-based XPCT

Intranasal insulin has been investigated as a metabolic intervention in Alzheimers disease (AD), but its effects across the brain-gut axis remain unclear. We combined multiscale synchrotron X-ray phase-contrast tomography (XPCT), amyloid-{beta} immunofluorescence, and fecal metaproteomics to examine structural and molecular responses in 5xFAD mice. Twenty-four mice representing wild-type and 5xFAD genotypes, saline and insulin conditions, and terminal ages of 2 and 6 months received insulin at 1 U/mouse/day for 5 weeks. Micro-XPCT visualized plaque-like deposits, nanoXPCT revealed their heterogeneous internal organization, and MOAB-2 labeling provided qualitative support for amyloid pathology. Detectable plaque burden was not lower after treatment: deposits occurred in two of three treated versus none of three untreated 5xFAD mice at 2 months, and treated animals had higher descriptive regional means at 6 months. In the ileum, insulin was associated with lower villus length and crypt depth in 6-month-old 5xFAD mice, whereas no significant differences were detected at 2 months. Fecal profiles retained broad taxonomic overlap but differed in functional annotations, with hydrolase-associated fractions of 81%, 92%, 96%, and 93% across untreated wild-type, treated wild-type, untreated 5xFAD, and treated 5xFAD conditions, respectively, within the selected peptide subset. Thus, lower ileal dimensions coexisted with non-reduced detectable brain plaque burden and a fecal protein profile distinct from untreated wild type. Together, these findings reveal contrasting central and peripheral patterns following intranasal insulin treatment in 5xFAD mice: no detectable reduction in cerebral plaque burden, alongside attenuation of selected ileal structural alterations and distinct fecal metaproteomic profiles. Although exploratory, the findings raise the possibility that intranasal insulin alters amyloid compartmentalization or plaque detectability rather than promoting amyloid clearance, while producing distinct peripheral remodeling.