Maternal exercise during lactation remodels obesity-associated mammary metabolism and milk fatty acids, enhancing offspring lipid oxidation

Maternal obesity alters breast milk composition in ways that may predispose infants to excess adiposity. While maternal exercise during lactation has been associated with favorable shifts in milk metabolites in humans, the mechanisms by which exercise remodels the mammary gland and milk lipid profile to influence offspring metabolism remain unclear. We developed a mouse model incorporating daily moderate treadmill exercise only during lactation, using lean (LN) and diet-induced obese (OB) dams, and leveraged indirect calorimetry, stable isotope tracer respirometry, and mammary epithelial cell (MEC) proteomics assays. Maternal obesity broadly remodeled the MEC proteome, decreasing enzymes of <i>de novo</i> fatty acid synthesis and altering lipid transport and oxidative pathways. These molecular adaptations in OB dams corresponded to higher milk triglyceride content and shifts in fatty acid composition, including suppressed medium chain fatty acids (MCFA). The exercise (EX) intervention during lactation reset MEC protein networks, enhancing protein translation and vesicle transport pathways, while decreasing fatty acid desaturation, relative to the sedentary (SED) group. In OB dams, the exercise intervention increased milk MCFA levels and partially corrected the pro-inflammatory omega-6 FA bias. Offspring suckling OB-EX dams exhibited enhanced <i>in vivo</i> fatty acid oxidation, partially rescuing obesity-associated impairments in metabolic fuel preference. Together, maternal exercise during lactation remodels mammary metabolism and milk fatty acid composition in obese dams, which in turn, enhances postnatal lipid oxidation. These findings highlight lactation as a modifiable window, wherein maternal activity influences milk composition and early life metabolism.