Fig. 4

PNE enhanced hypothalamic leptin signaling in offspring. a Plasma leptin determined in week 16 of the diets. b Hypothalamic POMC mRNA levels were determined in week 16 of the diets by reverse transcription quantitative PCR, normalized to β-actin mRNA and expressed relative to the STD-fed control group. The number of male offspring in each group in (a) and (b) was n = 7–9 originating from at least three different dams. c, d Leptin-induced nuclear STAT3 phosphorylation (P-STAT3) of hypothalamic arcuate nucleus neurons (c, d) and hypothalamic POMC neurons (d) determined in STD-fed mice by immunofluorescence using antibodies against STAT3-Tyr 705 phosphorylation and POMC polypeptide. P-STAT3 was detected using either a rabbit monoclonal (c) or mouse monoclonal antibody (d, left panel). Male offspring were fasted for 14 h, injected i.p. leptin (5 mg/kg), and sacrificed 30 min post injection. White arrows highlight several P-STAT3 reactive nuclei in the hypothalamic arcuate nucleus (ARC) (c, d, left panel), POMC positive neurons (d, middle panel), and POMC positive neurons with P-STAT3 reactive nuclei (d, right panel). Red arrows highlight neurons with cytoplasmic P-STAT3 immunoreactivity (c, right panel). 3 V refers to the third ventricle (c, d). The size bars represent 50 μM (c, d). e, f Quantification of leptin-induced nuclear STAT3 phosphorylation in hypothalamic arcuate neurons after staining with rabbit monoclonal P-STAT3 antibody (e) or mouse monoclonal P-STAT3 antibody (f) (n = 3 male offspring per group). g Quantification of P-STAT3 immunoreactive hypothalamic POMC neurons (n = 3 male offspring per group). Data were analyzed by two-way ANOVA followed by Bonferroni post-tests (*, p < 0.05; **, p < 0.01; ***, p < 0.001) (a, b) or by two-tailed t test (e, f). All data are expressed as mean ± SEM