Prolonged breastfeeding protects from obesity by hypothalamic action of hepatic FGF21

The study investigates whether the duration of breastfeeding (modeled by delayed weaning in rodents) can induce long-lasting reprogramming of energy balance that protects against obesity. Obesity has risen to pandemic levels and is influenced by environmental, genetic, and epigenetic factors, with early-life events implicated in neurodevelopmental and metabolic programming. While maternal diet during lactation and neonatal overfeeding have been linked to later obesity risk, evidence for a direct, durable effect of the offspring’s own suckling duration on adult energy homeostasis remains inconclusive. The authors hypothesize that prolonged suckling can durably enhance energy expenditure via brown adipose tissue (BAT) thermogenesis, mediated by peripheral signals—particularly hepatic FGF21—acting on hypothalamic circuits to counter diet-induced obesity (DIO).

Prior animal studies show that maternal high-fat diet (HFD) during lactation alters milk composition and predisposes offspring to adult obesity, glucose dysregulation, reduced BAT thermogenesis, and altered hypothalamic circuits. Epidemiological data on breastfeeding’s protective effects against later obesity are mixed due to confounding factors and study design limitations; some report protection while others find no association. Breast milk carries bioactive factors (e.g., leptin) that may program energy homeostasis, and breastfeeding duration correlates with epigenetic modifications of the leptin gene. FGF21 rises after birth with suckling and induces neonatal BAT thermogenesis; in adults, it improves glucose tolerance and promotes thermogenic activation. The authors build on these findings to test whether prolonged suckling elevates hepatic FGF21 and engages central pathways to yield durable anti-obesity effects.

- Animal models: Sprague-Dawley rats; delayed weaning (DW) at postnatal day 28 vs standard weaning (SW) at day 21. After weaning, males were fed chow diet (CD) or high-fat diet (HFD) to 18 weeks. A short-term variant matched HFD exposure (1 week) to control for the extra week of HFD in SW-HFD. - Transgenic mice: Liver-specific Fgf21 knockout (Alb-Cre; Fgf21 floxed); Drd2-Cre and Vgat-ires-Cre lines for cell-type-specific manipulations; controls were appropriate Cre-negative or scrambled vector groups. - Interventions and surgeries: Intracerebroventricular (ICV) cannulation; ICV injections of leptin (3 µg/rat) or recombinant human FGF21 (0.4 µg/animal). Stereotaxic AAV injections into the lateral hypothalamic area/zone incerta (LHA/ZI) to knock down D2 receptor (D2R) or FGFR1 in defined neuronal populations (Cre-dependent constructs). Liver-specific Fgf21 knockdown in rats via tail-vein lentiviral shRNA (shFgf21) vs shLuciferase control. Tanycyte-targeted FGFR1 knockdown via ICV AAV1/2 expressing Cre under hDio2 promoter combined with Cre-dependent shFgfr1. - Phenotyping: Weekly body weight, food intake, body composition by EchoMRI. Indirect calorimetry (energy expenditure, respiratory quotient, locomotor activity). Thermal physiology at thermoneutrality (30°C) and during cold exposure (4°C). Infrared thermal imaging of interscapular BAT. PET/CT 18F-FDG to quantify BAT glucose uptake (SUVmax) at room temperature and 4°C. - Metabolic tests: Glucose tolerance test and insulin tolerance test after fasting. Plasma lipids (triglycerides, cholesterol, NEFAs), leptin, and FGF21 by ELISA. - Molecular and histological analyses: Western blots for BAT thermogenic/lipolytic markers (UCP1, PGC1α, PPARγ, pHSL/HSL), hypothalamic signaling (pSTAT3/STAT3, pPI3K/PI3K, pAKT/AKT, pERK/ERK), LHA/ZI D2R and FGF21, orexin. Immunohistochemistry for UCP1 in BAT and WAT browning; Oil Red O staining for hepatic steatosis. Fluorescently labeled FGF21 injected via jugular vein or ICV to assess uptake by median eminence tanycytes; combined RNAscope FISH and immunofluorescence to evaluate Fgf21 transcripts and FGF21 protein in tanycytes. FACS isolation of LHA/ZI D2R GABA neurons for Fgfr1 mRNA detection. - Statistics: Normality and variance homogeneity testing (Kolmogorov–Smirnov, Levene). Group comparisons by one-way ANOVA with Tukey’s post hoc, Student’s t-test, Mann–Whitney U test for non-normal data, ANCOVA with body weight as covariate when appropriate. P<0.05 considered significant. Animal ethics approvals and controlled housing conditions described.

- Body weight and adiposity: Delayed weaning (DW) rats on HFD (DW-HFD) gained significantly less weight than standard weaning HFD (SW-HFD) despite similar food intake; fat mass was notably reduced while lean mass was unchanged. Energy expenditure was higher in DW-HFD in both light and dark phases without changes in locomotor activity or respiratory quotient. Plasma triglycerides, cholesterol, NEFAs, and leptin were reduced; glucose tolerance and insulin sensitivity improved. - Thermogenesis: DW-HFD had higher interscapular (iBAT) temperature at thermoneutrality and better maintenance of core and iBAT temperatures during cold exposure. BAT weight increased and lipid droplets were smaller. Thermogenic markers in BAT (UCP1, PGC1α, PPARγ, pHSL/HSL) were elevated. 18F-FDG PET/CT showed higher BAT SUVmax at room temperature (P=0.049) and at 4°C (P=0.028), indicating increased BAT glucose uptake. - WAT browning and liver: Subcutaneous WAT showed decreased lipid content, increased UCP1 immunolabelling and protein, elevated PGC1α, and higher pHSL/HSL ratio. Hepatic steatosis was ameliorated. Hepatic and plasma FGF21 levels were significantly increased in DW groups. - Leptin sensitivity: ICV leptin failed to reduce body weight or food intake in SW-HFD (leptin resistance) but significantly decreased both in DW-HFD. In DW-HFD, leptin activated hypothalamic signaling (increased pSTAT3/STAT3, pPI3K/PI3K, pAKT/AKT, pERK/ERK) in the mediobasal hypothalamus. - Causality of hepatic FGF21: Liver shRNA knockdown of Fgf21 in DW-HFD normalized hepatic and plasma FGF21 to SW-HFD levels and partially reversed benefits—body weight and fat mass increased, iBAT temperature fell, and BAT thermogenic markers (PGC1α, UCP1) and UCP1 immunolabelling decreased. Hepatic lipid accumulation and plasma triglycerides rose. LHA/ZI FGF21 and D2R protein levels declined toward SW-HFD levels, indicating liver-derived FGF21 drives central changes. - Central FGF21 action: ICV FGF21 in lean rats reduced body weight over 24 h independent of food intake, raised iBAT temperature for at least 24 h, increased BAT PGC1α and UCP1 proteins and UCP1 labelling, and elevated D2R protein in LHA/ZI. - Role of LHA/ZI D2R: AAV shD2r in LHA/ZI of DW-HFD rats reduced local D2R to SW-HFD levels and prevented DW-induced reductions in weight gain and adiposity, without changing feeding. It blunted increases in iBAT temperature and BAT UCP1/PGC1α and reversed improvements in liver steatosis and plasma triglycerides. - D2R requirement for FGF21 effects: In rats, LHA/ZI D2R knockdown abolished ICV FGF21-induced increases in iBAT temperature and BAT thermogenic markers. In D2r-Cre mice, Cre-dependent LHA/ZI shD2r prevented FGF21-induced c-Fos activation in D2R neurons and blocked FGF21-driven body weight loss, iBAT temperature rise, BAT UCP1 labelling, and protein increases. - FGFR1 on D2R neurons: Knocking down FGFR1 specifically in LHA/ZI D2R neurons (Cre-dependent AAV8-shFgfr1 in D2r-Cre mice) abolished FGF21-induced weight loss, BAT thermogenesis (iBAT temperature, UCP1 labelling, PGC1α/UCP1 proteins), indicating FGF21 acts directly via FGFR1 on these neurons. - D2R in GABA neurons: In Vgat-Cre mice, D2R knockdown selectively in LHA/ZI GABA neurons blocked FGF21-induced decreases in body weight and increases in iBAT temperature and BAT PGC1α/UCP1 without altering food intake, identifying D2R-expressing GABA neurons as effectors. - Tanycytic transport: Fluorescent FGF21 injected via jugular vein localized to median eminence tanycytes (not after ICV), supporting blood-to-brain tanycytic shuttling. DW-HFD rats showed increased FGF21 immunoreactivity in tanycytic processes but reduced tanycytic Fgf21 transcripts, consistent with enhanced uptake rather than local synthesis. Tanycyte-specific FGFR1 knockdown (AAV1/2-hDio2-iCre + shFgfr1) lowered iBAT temperature after cold exposure and reduced BAT UCP1/PGC1α, indicating FGFR1-dependent tanycytic transport is required for central thermogenic control by peripheral FGF21. - Control for HFD exposure duration: In a short-term model equalizing HFD exposure (1 week), DW still reduced body weight and increased iBAT temperature and BAT thermogenic markers, and at 4 weeks (before weight diverged) DW already increased iBAT temperature, indicating thermogenic changes precede weight differences.

The findings demonstrate that prolonged suckling durably reprograms energy balance by enhancing BAT thermogenesis and energy expenditure, conferring resistance to diet-induced obesity without altering food intake. Mechanistically, extended breastfeeding elevates hepatic FGF21 production in offspring, which persists into adulthood and reduces hepatic steatosis. Liver-derived FGF21 gains access to the hypothalamus via tanycyte-mediated transport at the median eminence and acts directly through FGFR1 on D2R-expressing GABAergic neurons in the LHA/ZI. Activation of this hypothalamic dopaminergic pathway increases BAT thermogenesis, improving glucose handling and lipid metabolism and restoring leptin sensitivity. Loss-of-function experiments at key nodes—hepatic Fgf21, LHA/ZI D2R, FGFR1 on D2R neurons, and tanycytic FGFR1—each abrogate the thermogenic and anti-obesity phenotype, establishing causality across the liver–tanycyte–hypothalamus axis. These results provide a coherent mechanism linking an early-life nutritional experience (breastfeeding duration) to long-lasting central circuit remodeling that mitigates metabolic disease risk.

This work establishes that delayed weaning (a model of prolonged breastfeeding) protects against adult diet-induced obesity by increasing BAT thermogenesis and WAT browning, improving lipid and glucose homeostasis, and restoring leptin sensitivity independently of food intake. The protection is mediated by sustained increases in hepatic FGF21 that, via tanycytic transport, activate FGFR1 on D2R-expressing GABA neurons in the LHA/ZI to drive thermogenesis. The study highlights the liver–tanycyte–hypothalamus circuit as a targetable pathway for metabolic disease. Future research should: (1) define the epigenetic and molecular mechanisms sustaining elevated hepatic FGF21 after prolonged suckling; (2) map downstream autonomic outputs from LHA/ZI D2R neurons to BAT; (3) assess sex differences and the role of early-life diet composition; and (4) translate to humans by evaluating whether breastfeeding duration correlates with circulating FGF21 trajectories, BAT activity, and hypothalamic markers, while accounting for confounders.

- Translation to humans is uncertain due to species differences and multiple life-course confounders in epidemiology; the study uses controlled animal models. - Only male offspring were studied after weaning; potential sex-specific effects were not addressed. - Viral vector approaches (lentivirus/AAV) can have off-target, immune, and clearance limitations; although scramble controls were used, residual sequence-specific off-target effects cannot be fully excluded. - DW vs SW had a nominal difference in HFD exposure; although a matched short-term experiment and re-analysis by diet weeks support conclusions, residual confounding cannot be entirely excluded. - Measurements of central FGF21 action focus on LHA/ZI D2R GABA neurons; other brain regions or cell types may contribute to FGF21’s broader effects (e.g., macronutrient preference), which were not fully explored.