(P16-20-26) Milk Consumption May Normalize Redox and Methylation Homeostasis in FMR1 KO Mice Independent of β-Casein Subtype

Objectives: Given altered opioid peptide metabolism and redox imbalance reported in autism spectrum disorder, this study aimed to determine whether A1 and A2 containing milks differentially modulate behavioral outcomes, inflammatory markers, and redox/methylation metabolism in fragile X mental retardation 1 (FMR1) gene knockout mice, a model exhibiting autism-like phenotypes. β-casein variants in bovine milk (A1 and A2) differ in digestion-derived peptides; A1 β-casein produces β-casomorphin-7 (BCM-7), an opioid peptide linked to gut permeability, immune activation, and neurobehavioral regulation, while A2 β-casein does not.

Methods: Male B6.129P2-Fmr1^tm1Cgr/J (FMR1 KO, n = 45) and C57BL/6J (B6, n = 15) mice were maintained on a casein-free diet for 19-26 days prior to intervention. At 7-8 weeks of age, FMR1 KO mice were randomized to receive saline, conventional A1/A2 milk, or A2/A2 milk (10 mL/kg/day) via oral gavage for 30 consecutive days (n = 15/group). B6 controls received saline (10 mL/kg/day) for the same duration. Following the intervention, mice underwent the three-chamber social preference and open-field tests. Blood plasma along with brain, duodenum, jejunum, and ileum tissues were collected for analysis.

Results: Redox and methylation metabolite analyses revealed significant genotype-associated alterations in the FMR1 KO saline mice compared with B6 controls, including reduced GSH/GSSG (p=0.01) and SAM/SAH (p < 0.001) ratios and elevated methionine level (p < 0.0001). Both milk-treated FMR1 KO groups exhibited normalization of these metabolite concentrations toward B6 control levels, with no significant differences between the two milk types. In the open-field test, FMR1 KO mice on saline exhibited greater locomotor activity compared with B6 mice, and milk consumption did not alter this activity. Behavior during the three-chamber test and concentrations of the plasma cytokines (TNF-α, MCP-1, IL-1β, IL-10, and IL-6) did not differ between groups. Analyses of BCM-7 content and gastrointestinal and brain inflammatory cytokines are ongoing.

Conclusions: These results indicate that milk consumption may normalize redox and methylation homeostasis in FMR1 KO mice independent of β-casein subtype while behavioral and circulatory inflammatory outcomes were unaffected.

Funding Sources: Gift funds from a2 milk company