Fig 2: Ob-R, kisspeptin, and GPR54 protein expression in the testis (A) showed the results of Ob-R in the testis: the depict representative findings and the results of the semi-quantitative measurement in the control, DEHP100, DIO, DIO + DEHP30, DIO + DEHP100, and DIO + DEHP300 groups. B showed the results of kisspeptin in the testis: the depict representative findings and the results of the semi-quantitative measurement in the control, DEHP100, DIO, DIO + DEHP30, DIO + DEHP100, and DIO + DEHP300 groups. C showed the results of GPR54 in the testis: the depict representative findings and the results of the semi-quantitative measurement in the control, DEHP, DIO, DIO + DEHP30, DIO + DEHP100, and DIO + DEHP300 groups. Each bar represents mean ± SE. n = 4. (a) p < 0.05 denote statistically significant compared with control group; (b) p < 0.05 denote statistically significant compared with DIO group; (c) p < 0.05 denote statistically significant compared with DIO + DEHP30 group; (d) p < 0.05 denote statistically significant compared with DEHP100 group. DEHP = di-(2-ethylhexyl) phthalate; DIO = diet-induced obesity.

Fig 3: LRP6 Mediates the Uptake of oxPLs by MSCs. a–d Fluorescence imaging of the binding and uptake of DiI-nLDL and DiI-oxLDL (dissolved in PBS) by MSCs. Bone marrow MSCs isolated from GFP transgenic mice were transfected with either irrelevant control siRNA (siControl) (a, c) or LRP6 siRNA (siLRP6) (b, d). 2 days later, the cells were incubated with DiI-nLDL (a, b) or DiI-oxLDL (c, d) for 2 h at 4 ?, and were then incubated at 37 ? for 0 min, 10 min, or 30 min. Cells were washed, fixed, and analyzed by fluorescence microscopy. (Scale bar: 10 µm). e Mice were injected with DiI-nLDL or DiI-oxLDL (dissolved in PBS) via tail vein at a dose of 20 µg per mouse. 4 h after injection, Mice were killed at 4 h after injection, and the bone marrow cells were subjected to flow cytometry analysis. The percentages of the DiI-nLDL or DiI-oxLDL uptake by LepR+CD45–CD31–Ter119- MSCs are shown in the lower panel. f, g LRP6+/+::LepR-Cre and LRP6f/+::LepR-Cre mice were injected with DiI-oxLDL via tail vein at a dose of 20 µg per mouse. 4 h after injection, mice were killed, and bone marrow cells were subjected to flow cytometry analysis. Representative images of the percentage of LRP6+ cells are shown in f. The percentages of the DiI-oxLDL uptake by LepR+CD45–CD31–Ter119- cells are shown in g. n = 5, data are represented as mean ± s.e.m

Fig 4: Cell Surface LRP6 Is Reduced in Bone Marrow MSCs under Microenvironment of High oxPLs. a–c Representative images (a) and the quantified CFU-F frequency (b), and CFU-Ob frequency (c) of bone marrow MSCs from 10-week-old C57BL/6 mice fed Western high fat diets (HFD) or standard chow diets (CHD) for 4 weeks. d Bone marrow MSCs from 10-week-old C57BL/6 mice fed HFD or CHD for 4 weeks were incubated with osteogenic medium for 7 days. mRNA expressions of RUNX2 and osteocalcin (OCN) were detected by Real-time PCR analysis. n = 5 mice per treatment group from six independent experiments, data are represented as mean ± s.e.m. *p < 0.05, vs. CHD group, as determined by Student’s t-tests. e–h Representative images of the flow cytometry analysis (e) and the percentage of LRP6+ (f), CD36+ cells (g), and LOX-1 (h) in LepR+CD45–CD31–Ter119- cell population from 10-week-old C57BL/6 mice fed Western high fat diets (HFD) or standard chow diets (CHD) for 2 weeks. n = 5, data are represented as mean ± s.e.m. *p < 0.01 vs. CHD group, as determined by Student’s t-tests. i–l Representative images of the flow cytometry analysis (i) and the percentage of LRP6+ (j), CD36+ cells (k), and LOX-1 (l) in CD11b+ cell population from 10-week-old C57BL/6 mice fed Western high fat diets (HFD) or standard chow diets (CHD) for 2 weeks. n = 5, data are represented as mean ± s.e.m. *p < 0.01 vs. CHD group, as determined by Student’s t-tests. m–o 10-week-old C57BL/6 mice fed CHD or HFD together with D-4F peptide (HFD + D-4F) or scrambled D-4F peptide (HFD + SD-4F) for 2 weeks. The peptides were administered orally in drinking water at 0.4 mg·mL-1, equivalent to 1.6 mg/d. Flow cytometry analysis of LRP6+ cells in bone marrow LepR+CD45–CD31–Ter119- MSCs (i). Representative histograms showing relative intensity of LRP6 antibody-conjugated fluorescence in the three groups (j). Quantitative analysis of the relative fluorescence intensity in the three groups (k). n = 5, data are represented as mean ± s.e.m. *p < 0.01 vs. CHD group, #p < 0.01, vs. HFD + SD-4F group, as determined by Student’s t-tests

Fig 5: The neonatal leptin surge stimulates ciliogenesis and neural circuit formation in the hypothalamus.a Cilia (AC3) staining in the hypothalamic ARH and hippocampal dentate gyrus of C57 neonates at P14 that received either saline or leptin antagonist SHLA injections from P4 to P13. The average lengths of 100 cilia and the ciliated cell percentage in each area per mouse are presented (n = 5 for hypothalamus, n = 4 for hippocampus). Scale bars: 20 µm. b Cilia staining in the hypothalamus (Hypo) and hippocampus (Hippo) of ob/+ mice, ob/ob mice, and ob/ob mice with leptin treatment (P10–P14) (n = 5). The average lengths of 100 cilia in each area per mouse are presented. Scale bars: 20 µm. c Upper panel: cilia (AC3) and POMC (ß-END) costaining in the hypothalamic ARH in 7-week-old ob/+ mice, ob/ob mice, and ob/ob mice with adulthood leptin replacement (10 mg/kg/day for 7 days; n = 5). Lower panel: axonal projections of POMC neurons in the three groups (n = 4). Scale bars: 50 µm. d Ciliogenesis in the ARH and POMC axonal projection to the PVH in ob/ob neonates injected with saline, leptin alone, or leptin + shIFT88-GFP-AAV (n = 4 for AAV on-target group, n = 5 for the other 3 groups). The mice with off-target AAV injection were used as a control. Scale bars: 50 µm. e AC3 (cilia), functional leptin receptor (LepRb), GFP (AAV transfection), and DAPI costaining in the ARH of ob/ob neonates injected with saline, leptin, or leptin + shIFT88-GFP-AAV. The percentages of ARH cells and AAV-infected cells with periciliary leptin receptor accumulation were counted (n = 5 for the ob/ob-saline group and n = 4 for the other 2 groups). Scale bars: 10 µm. Data values are presented as mean ± SEM. Statistics were performed using two-sided Student’s t test (a) and one-sided one-way ANOVA (b–e) followed by post hoc LSD test. **p < 0.01, ***p < 0.001 between the indicated groups. ns not significant.