**Background:** Nutritional adversities during critical developmental windows (gestation and lactation) increase the risk of obesity and metabolic disease across the lifespan—a phenomenon known as metabolic programming. The endocannabinoid system (ECS), which regulates food intake, fat preference, and lipogenesis, is overactivated in obesity and plays a key role in neurodevelopment. Endocannabinoids such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG) have been identified in human milk, but their presence in rodent milk and the effects of maternal diet on milk endocannabinoids were unknown. The authors hypothesized that maternal high-fat (HF) diet would increase milk endocannabinoid levels, programming cannabinoid and dopamine signaling in the nucleus accumbens (NAc) and altering food preference in offspring.
**Methods:** Female Wistar rats were assigned to a control diet (C; 10.9% fat, 3.34 kcal/g) or HF diet (28.7% fat, 3.65 kcal/g) for 8 weeks before mating and throughout gestation and lactation. Milk samples were collected at postnatal day 11 (mid-lactation) and day 21 (late lactation) for quantification of AEA and 2-AG by liquid chromatography–mass spectrometry, and fatty acid profile by gas chromatography–mass spectrometry. At weaning (postnatal day 21), male and female offspring were euthanized for adiposity assessment (visceral and subcutaneous white adipose tissue), serum hormonal profiling (leptin, insulin, PYY, GLP-1, glucose, triglycerides, cholesterol), and analysis of ECS and dopamine signaling proteins in the NAc by Western blotting. A separate cohort of offspring received control diet after weaning and underwent a food preference test at adolescence (postnatal day 45), with simultaneous access to control, HF, and high-sugar diets for 24 hours.
**Key Results:** Maternal HF diet reduced milk AEA at postnatal day 11 (−71%, p<0.05) and day 21 (−72%, p<0.05), and reduced milk 2-AG at day 11 (−87%, p=0.10) and day 21 (−62%, p=0.054). Maternal HF diet also decreased milk polyunsaturated fatty acids at mid-lactation, including eicosapentanoic acid (−30%, p<0.05), linoleic acid (−51%, p<0.05), arachidonic acid (−40%, p<0.05), and docosatetraenoic acid (−55%, p<0.05), while increasing saturated fatty acids at late lactation (2–3-fold, p<0.05). In the mammary gland, maternal HF diet increased CB2 receptor content (+1.6-fold, p<0.05) but did not alter CB1, FAAH, MAGL, NAPE-PLD, or DAGL protein levels.
Offspring from HF dams showed increased adiposity at weaning: male offspring had 2.1-fold higher visceral and 95% higher subcutaneous fat (p<0.05); female offspring had 2.7-fold higher visceral and 2.1-fold higher subcutaneous fat (p<0.05). Maternal HF diet increased glycemia (male: +24%, p<0.05; female: +14%, p<0.05) and insulinemia (male: +3.6-fold, p<0.05) at weaning, with a significant maternal diet effect for insulin (p<0.05).
In the NAc, maternal HF diet increased CB1 (+56%, p=0.06) and CB2 (+67.6%, p<0.05) in male offspring, with decreased FAAH (−56.1%, p<0.05). In female HF offspring, MAGL was decreased (−50%, p<0.05) with no changes in CB1, CB2, or FAAH. Dopamine signaling showed sex-specific changes: male HF offspring had increased D1R, D2R, and DAT but decreased DARPP-32 (−46.3%, p<0.05); female HF offspring had decreased D2R and DAT but increased DARPP-32 (+71.2%, p<0.05). Two-way ANOVA revealed significant interaction effects (maternal diet × offspring sex) for most ECS and dopamine markers (p<0.05).
In the food preference test at adolescence, male HF offspring showed 2.4-fold higher preference for HF diet (p<0.05) compared to controls, with no differences in preference for control or high-sugar diets. Female HF offspring showed no alterations in food preference.
**Clinical Implications:** This study demonstrates for the first time the presence of endocannabinoids in rat breast milk and shows that maternal HF diet reduces milk AEA and 2-AG levels, contrary to the authors' hypothesis. The reduced endocannabinoid exposure during lactation may induce adaptive, sex-dependent changes in cannabinoid-dopamine crosstalk within the developing NAc, contributing to altered neurodevelopment and programming of fat preference in male offspring. These findings highlight the importance of maternal diet quality during lactation for offspring neurobehavioral development and obesity risk, and suggest that the endocannabinoid content of breast milk may be a modifiable factor influencing long-term metabolic health. The sex-specific effects underscore the need to consider sex as a biological variable in developmental programming research.