Alternate-day fasting delays pubertal development in normal-weight mice but prevents high-fat diet-induced obesity and precocious puberty

The study examines whether alternate-day fasting (ADF), a lifestyle-based intervention, can modulate pubertal timing and growth under normal nutrition and in the context of diet-induced obesity. Precocious puberty is increasing globally and is associated with overnutrition and childhood overweight/obesity, contributing to adverse cardiometabolic and oncologic outcomes and reduced adult height. Puberty results from early activation of the hypothalamic-pituitary-gonadal (HPG) axis, and prior animal work shows postnatal high-fat diet (HFD) can induce obesity and advance puberty, whereas perinatal undernutrition delays puberty. Existing treatments (e.g., GnRH agonists) can be effective but are invasive, costly, and carry side effects. Intermittent fasting has been reported to inhibit the HPG axis and improve weight in obesity. The research question is whether ADF after weaning affects pubertal onset, growth, and related hormones in normal-weight female mice and whether it can prevent HFD-induced obesity and precocious puberty.

Prior studies link childhood obesity to earlier puberty, especially in girls, and associate early puberty with increased cardiovascular, metabolic, cancer risks, and reduced adult height. Animal studies by the authors have shown postnatal HFD induces obesity and precocious puberty in mice. Undernutrition reduces body weight and delays puberty. Intermittent fasting can inhibit the HPG axis in rats and improve adiposity and metabolic parameters in obese humans. Leptin is a permissive factor for pubertal initiation and correlates with body fat; higher adiposity and leptin are associated with earlier menarche. GH/IGF-1 axis mediates linear growth responses to nutrition, but its role in pubertal timing is less clear. These findings motivated testing ADF as a non-pharmacologic approach to manage obesity and precocious puberty.

Animals: Female C57BL/6J mice (GEMPHARMATECH, Shanghai) housed under standard conditions (12 h light:12 h dark; lights on 6:00 A.M.) with ad libitum access to food/water. Breeding pairs at 12 weeks; day of birth marked P1; only female pups used. Study approved by Zhejiang University Animal Advisory Committee. Diets: High-fat diet (HFD; D12492, 60% kcal fat, 20% protein, 20% carbohydrates) and standard control chow (1010097; 15% kcal fat, 24% protein, 61% carbohydrates). Experimental design: Dams on control chow before/during pregnancy; at parturition, dams assigned to chow or HFD; offspring post-weaning at P21 continued maternal diet. Groups: control chow (Cntrl), chow+ADF, HFD, HFD+ADF. ADF protocols followed published methods with adjustments: 18 h ADF (fast from 5 P.M. to 11 A.M. on alternate days) or 24 h ADF (fasting 24 h starting 5 P.M. on alternate days). For HFD+ADF group with blood collection at 11 A.M. on P34, fasting started 11 A.M. the prior day. VO typically ~P33 in controls; 18 h ADF delayed VO to ~P40; analyses in chow-fed mice focused on P34 and P40. Litter size 6–7. Group sizes varied by parameter (n≈6–20 per group); each data point represents an individual mouse. Exclusion criteria: weak/sick mice or hemolyzed samples. Outcomes: Body weight, body length (nose-to-anus), uterine and ovarian weights. Puberty onset assessed by vaginal opening (VO) monitored daily from P21. Blood sampling and assays: Plasma collected at 11 A.M. (cardiac puncture; 3000×g, 15 min; stored at −80°C). ELISAs (HAKATA, China) measured LH (HZ-030192; sensitivity 1 mIU/mL), FSH (HZ-030195; 1 mIU/mL), estradiol E2 (HZ-030188; 1 pmol/mL), insulin (HZ-030681; 1 mIU/L), leptin (HZ-030555; 1 ng/mL), growth hormone GH (HZ-030777; 1 ng/mL), IGF-1 (HR-010992; 1 ng/mL), per manufacturer protocols. GH treatment: In chow-fed 18 h ADF mice, recombinant human GH (rhGH; GenSci) administered subcutaneously daily in afternoon (3–4 P.M.) at 2 IU/kg/day or 4 IU/kg/day; vehicle group received saline. Volume 10 µL/g body weight. Statistics: Data shown as mean±SEM. Outliers removed by Iterative Grubbs test. Normality checked (Kolmogorov–Smirnov, Shapiro–Wilk). For two groups with Gaussian distribution: unpaired two-tailed t-test; for non-Gaussian or unequal variances: Mann–Whitney. Multiple groups: one-way ANOVA with Tukey’s post hoc for Gaussian; otherwise Brown–Forsythe/Bartlett’s to assess variances, followed by Kruskal–Wallis with Dunn’s post hoc. GraphPad Prism v8; significance p<0.05.

- In chow-fed normal-weight mice, 18 h ADF delayed puberty (VO) markedly (P<0.0001) and reduced body weight (P<0.0001), body length (P<0.0001), uterine weight (P=0.0006 to <0.0001), and ovarian weight (P=0.0224 to <0.0001). In controls, body metrics increased from P34 to P40 (e.g., BW P=0.0005; length P=0.0006; uterine weight P=0.0028), but not in ADF mice. - Sex hormones under 18 h ADF (chow-fed): At P34, FSH (P=0.0059) and E2 (P<0.0001) were reduced vs controls; at P40, no significant differences for FSH (P=0.4479) or E2 (P=0.1262). LH: similar at P34 (P=0.6053) but elevated at P40 with ADF (P=0.0053). Within-group changes: Cntrl FSH and E2 decreased P34→P40 (P=0.0147; P<0.0001), while ADF maintained comparable levels (FSH P=0.2077; E2 P=0.8665). LH increased P34→P40 in ADF (P=0.0296). - Metabolic/growth hormones under 18 h ADF (chow-fed): Leptin and insulin showed no differences between ADF and control at P34 or P40; leptin decreased P34→P40 in both groups (P<0.0001; P=0.0004). GH decreased with ADF at P34 (P=0.0317) and increased at P40 (P=0.0075) vs control; GH declined P34→P40 in controls (P<0.0001) but not in ADF (P=0.8479). IGF-1 trended lower at P34 (P=0.0939), comparable at P40 (P=0.2523); IGF-1 decreased P34→P40 in both groups (P=0.0013; P<0.0001). - In HFD-fed mice, 18 h ADF did not prevent precocious puberty (VO; P=0.2398) but attenuated increased body weight (P=0.0408) and length (P=0.0477); uterine weight not rescued (P=0.9550). Hormones at P34: LH unchanged (P=0.8541); FSH partially restored (P=0.0132); E2 showed a trend (P=0.0777). GH and IGF-1 unaffected by HFD or 18 h ADF (P=0.2622; P=0.1534). - In HFD-fed mice, 24 h ADF prevented precocious puberty (VO; P<0.0001) and counteracted increases in body weight (P=0.0005) and length (P<0.0001). Uterine weight increase was prevented (P=0.0003); ovarian weight unaffected by HFD or 24 h ADF (P>0.9999). LH unchanged (P=0.9927). FSH and E2 were normalized with 24 h ADF (both P<0.0001). GH, IGF-1, and insulin were not altered by HFD or 24 h ADF; 24 h ADF prevented HFD-induced leptin elevation (P=0.0020). - Comparison of 24 h vs 18 h ADF in HFD-fed mice (Supplementary): 24 h ADF more effectively reduced uterine weight (P=0.0005), lowered LH (P<0.0001), and increased FSH (P=0.0001) and E2 (P<0.0001) vs HFD; 18 h ADF had minimal effects except a modest FSH increase. - GH replacement during 18 h ADF (chow-fed) rescued reduced body weight (P=0.0053) and length (P=0.0008 and P<0.0001 for doses) but did not prevent delayed puberty (P=0.7446 and >0.9999) nor reduced uterine/ovarian weights (both >0.9999).

The findings demonstrate that the peripubertal period is highly sensitive to energy status. In normal-weight mice, 18 h ADF delays pubertal onset and reduces somatic and reproductive organ growth, associated with lowered FSH/E2 and altered GH/IGF-1, indicating that sex steroid suppression is a key mechanism for delayed puberty, while reduced GH/IGF-1 contributes to impaired linear growth. GH supplementation restored growth but not puberty timing or reproductive organ weights, supporting distinct regulatory pathways for growth vs pubertal activation under energy restriction. In HFD-induced obesity, pubertal advancement occurred alongside increased leptin and altered gonadotropins, without changes in GH/IGF-1, indicating that leptin and sex steroids rather than GH/IGF-1 drive accelerated growth and precocious puberty. While 18 h ADF mitigated obesity metrics, it was insufficient to prevent precocious puberty; extending fasting to 24 h normalized uterine weight and FSH/E2 and prevented precocious puberty, also suppressing leptin elevations. Thus, ADF, particularly 24 h ADF, can counteract HFD-driven endocrine alterations underlying obesity and early puberty. These results highlight ADF as a potential non-invasive strategy to manage diet-induced obesity and pubertal timing, while cautioning that in normal-weight individuals, prolonged fasting may inadvertently delay puberty and impair growth.

ADF delays puberty in normal-weight female mice by reducing sex hormone levels and attenuating GH/IGF-1, leading to reduced growth; GH supplementation restores growth but not puberty timing. HFD induces obesity and precocious puberty with elevated leptin and altered gonadotropins, effects that 24 h ADF prevents, normalizing body growth and FSH/E2 without altering GH/IGF-1 or insulin. Although 18 h ADF improves obesity metrics in HFD-fed mice, it does not prevent precocious puberty. ADF, especially 24 h ADF, emerges as a promising lifestyle-based approach to manage diet-induced childhood obesity and associated early puberty, but translation to clinical practice will require further mechanistic studies and clinical trials.

The study was conducted in mice, limiting direct generalizability to humans. Hormonal and morphometric assessments were performed at specific time points (primarily P34 and P40), which may not capture full temporal dynamics. The work did not evaluate whether ADF corrects HFD-induced hypothalamic metabolic inflammation, feeding circuitry alterations, or glucose intolerance. While associations with leptin and sex steroids were observed, causal pathways (e.g., leptin signaling to HPG axis) were not directly manipulated. Sample sizes varied by outcome, and some comparisons showed variance heterogeneity requiring nonparametric tests.