Prolonged breastfeeding protects from obesity by hypothalamic action of hepatic FGF21

Obesity has reached pandemic levels, influenced by environmental, genetic, and epigenetic factors. Neonatal events are believed to play a significant role in neurodevelopment and later adult behavior, suggesting that early-life determinants may contribute to the rise in obesity. Maternal diet and neonatal feeding are key determinants. Studies have shown that maternal high-fat diets during lactation predispose offspring to obesity and impaired glucose homeostasis due to impaired white adipose tissue (WAT) function, inhibited brown adipose tissue (BAT) thermogenesis, and damaged hypothalamic circuits. While neonatal overfeeding's impact on long-term metabolic outcomes is suggested, the offspring's feeding/suckling influence on energy balance reprogramming remains inconclusive. Epidemiological studies show a potential protective effect of breastfeeding against obesity, but these studies are often limited by sample size and confounding factors. Breast milk contains bioactive factors modulating energy homeostasis, such as leptin, which is linked to postnatal programming and epigenetic alterations in the leptin gene. This study investigated whether breastfeeding's impact on energy homeostasis is long-lasting and the underlying molecular mechanisms. A rat model of prolonged breastfeeding (delayed weaning) followed by chow diet (CD) or high-fat diet (HFD) feeding was used, along with in-depth metabolic phenotyping and studies in transgenic mice to investigate the peripheral and central effects of prolonged suckling on HFD-induced long-term changes.

Extensive research has explored the impact of maternal nutrition during lactation on offspring obesity programming. Maternal high-fat diets (HFD) during lactation alter milk composition, leading to obesity and impaired glucose homeostasis in the offspring. This is linked to impaired WAT function, reduced BAT thermogenesis, and disrupted hypothalamic circuits. Conversely, studies on the impact of the infant's own feeding (specifically, breastfeeding duration) on long-term energy balance reprogramming have yielded mixed results. Some epidemiological studies suggested a protective effect of breastfeeding against childhood and adult obesity, while others found no correlation. Breast milk contains several bioactive factors that could modulate energy homeostasis. For example, leptin in breast milk is associated with healthy adult phenotypes, and breastfeeding duration is linked to epigenetic changes in the leptin gene. The current understanding lacked a clear picture of the long-term effects and underlying molecular mechanisms of prolonged breastfeeding on metabolic reprogramming.

The study used a rat model with prolonged suckling (delayed weaning to postnatal day 28 compared to standard weaning at day 21) followed by either CD or HFD feeding until 18 weeks of age. Body weight, body composition (NMR), energy expenditure, respiratory quotient, locomotor activity, food intake, glucose tolerance, and insulin sensitivity were assessed. The effect of leptin administration was also evaluated. BAT thermogenesis was examined through interscapular temperature measurement, histological analysis (lipid droplet size, UCP1 immunolabelling), and protein levels of UCP1, PGC1α, PPARγ, and FGF21. Glucose uptake in BAT was measured using 18F-FDG PET/CT. The role of FGF21 was investigated by hepatic FGF21 knockdown using lentiviral vectors encoding shRNA against FGF21. The hypothalamic mechanisms were explored by measuring FGF21 and D2 receptor (D2R) protein levels in the lateral hypothalamic area (LHA) and zona incerta (ZI). The effects of centrally administered FGF21 were also evaluated in lean rats. The role of D2R in the LHA/ZI was further investigated by D2R knockdown using adeno-associated viruses (AAVs). Transgenic mice lacking liver FGF21 and mice with D2R knockdown in the LHA/ZI were used to confirm findings. The route of FGF21 entry into the hypothalamus was studied using fluorescently labeled FGF21 and by inhibiting FGFR1 expression in tanycytes using AAVs. Finally, the specific cell population mediating FGF21 effects (D2R-expressing GABAergic neurons) was identified using FACS to isolate these neurons for mRNA expression analysis of FGFR1. Statistical analysis included ANOVA, t-tests, and Mann-Whitney U tests.

Prolonged suckling significantly reduced high-fat diet-induced weight gain and fat mass in rats, with increased energy expenditure but no change in food intake. Delayed-weaning rats showed enhanced BAT thermogenesis, indicated by higher interscapular temperature, smaller lipid droplets in BAT, increased UCP1 expression, and increased glucose uptake in BAT. Prolonged suckling also improved leptin sensitivity in HFD-fed rats. Hepatic FGF21 levels were significantly increased in delayed-weaning rats, and hepatic FGF21 knockdown blunted the beneficial effects of prolonged suckling on weight loss, adiposity, and BAT thermogenesis. This suggests that increased hepatic FGF21 production is causally linked to the protective effects of prolonged suckling. Prolonged suckling increased FGF21 and D2R levels in the LHA/ZI. Central administration of FGF21 mimicked the effects of prolonged suckling, increasing BAT thermogenesis and D2R expression in the LHA/ZI. D2R knockdown in the LHA/ZI reversed the protective effects of prolonged suckling against DIO. FGF21 was found to be shuttled into the hypothalamus by tanycytes. Further experiments showed that FGF21 acts directly on D2R-expressing GABAergic neurons in the LHA/ZI via its receptor FGFR1 to mediate its metabolic effects.

This study provides strong evidence that prolonged breastfeeding protects against diet-induced obesity through a mechanism involving increased hepatic FGF21 production, which acts centrally on the hypothalamus. The increased FGF21 levels, likely transported to the brain by tanycytes, activate specific GABAergic neurons expressing D2R in the LHA/ZI. This activation leads to enhanced BAT thermogenesis and energy expenditure, ultimately reducing weight gain and improving metabolic parameters. This finding highlights a novel link between a mother-infant interaction during a critical postnatal period and the long-term programming of hypothalamic circuitry for metabolic control. The study addresses the long-standing question of breastfeeding's impact on obesity by providing a mechanistic explanation for its potential protective effects. The animal model used minimizes confounding factors, providing clearer insights than epidemiological studies. The results support the growing recognition of early-life events' importance in shaping lifelong metabolic health. The findings have implications for developing potential interventions targeting hepatic FGF21 production or hypothalamic D2R signaling to improve metabolic health.

This research demonstrates that prolonged breastfeeding protects against diet-induced obesity by increasing hepatic FGF21, which, via tanycytic transport, acts on hypothalamic D2R-expressing GABAergic neurons in the LHA/ZI, leading to enhanced BAT thermogenesis. This provides mechanistic insight into breastfeeding's long-term metabolic benefits. Future research could focus on translating these findings into human interventions, such as exploring the potential of FGF21 analogs or targeted therapies for hypothalamic D2R signaling to improve metabolic outcomes in individuals at risk of obesity.

The study was conducted in rats and mice, and the findings may not directly translate to humans. The use of viral vectors for gene knockdown has limitations, such as potential off-target effects and immune responses. Although controls were used to minimize off-target effects, these risks remain. Further research is necessary to confirm these findings in humans and to determine the clinical relevance of these findings and potential of FGF21 analogs in human populations.