Fig 2: Metformin inhibits tumor necrosis factor-alpha (TNF-a) production, alters AMPK/PI3K/mTOR phosphorylation and induces metabolic reprogramming in pathological B cells from dehydroepiandrosterone (DHEA)-induced polycystic ovary syndrome (PCOS) mice.(A) Percentage of TNF-a+ cells in splenic CD19+ B cells by flow cytometric analysis (n=5–9 mice per group). (B) Western blot of representative images and quantification analysis of the ratios of phosphorylated (P-) AMPK (S487)/AMPK, P-PI3K p85 (Tyr458)/PI3K p85, and P-mTOR (S2481)/mTOR in splenic B cells from DHEA-induced PCOS mice (n=2 mice per group). (C) The heat-map analysis of energy metabolites in splenic B cells was analyzed by mass spectrometry (n=3 mice per group). (D) Differential energy metabolites were quantitatively analyzed by mass spectrometry (n=3 mice per group). For (A), p values were determined by Kruskal-Wallis test followed by Dunn’s post-hoc test and data are presented as medians with interquartile ranges. For (D), p values were determined by one-way ANOVA with Bonferroni’s multiple comparison post-hoc test and data are presented as means ± SEM. *p<0.05; **p<0.01. Figure 8—source data 1.Data points for graphs in Figure 8.

Fig 3: Metformin inhibits mechanistic target of rapamycin (mTOR) phosphorylation in pathological B cells.(A–D) Western blot of representative images and quantification analysis of the ratios of phosphorylated (P-) AMPK (S487)/AMPK, P-PI3K p85 (Tyr458)/PI3K p85, and P-mTOR (S2481)/mTOR in B cells isolated from peripheral blood (PB) of women with polycystic ovary syndrome (PCOS) (n=3 per group). (E–H) Western blot of representative images and quantification analysis of the ratios of P-AMPK (S487)/AMPK, P-PI3K p85 (Tyr458)/PI3K p85, and P-mTOR (S2481)/mTOR in stimulated peripheral blood B cells from women with PCOS (n=3 per group) with or without metformin (12.5 mM, Met) for 48 hr in vitro. (I–J) Western blot of representative images and quantification analysis of the ratio of P-mTOR (S2481)/mTOR in B cells isolated from PB of women with PCOS, before (pre) and after (post) treatment with metformin for one month (n=3 per group). p values were determined by two-tailed Student’s t-test and data are presented as means ± SEM. *p<0.05; **p<0.01. Figure 3—source data 1.Data points for graphs in Figure 3.

Fig 4: Effects of peripubertal exposure to diet containing 2% OA on the serum level of IGF-1 and E2, expression of CD36 and Cyclin D1, and activation of PI3K/Akt in the right side of the mammary gland of mice(A–B) Effects of dietary 2% OA on the serum levels of IGF-1 (A) and E2 (B) (n = 10). (C) Western blot analysis of CD36, p-PI3K, PI3K, p-Akt, Akt and cyclin D1 in the mammary gland of pubertal mice (n = 6). ß-actin was used as the loading control. (D) Mean ± SEM of immunoblotting bands of CD36, p-PI3K/PI3K, p-Akt/Akt and Cyclin D1, and the intensities of the bands were expressed as the arbitrary units. **P < 0.01 versus the Control group.

Fig 5: Immunohistochemical examination of PI3K expression in the ovaries of each group. Negative control staining with serum instead of PI3K primary antibody in (A) blank control, (B) vehicle control, (C) PCOS and (D) DMBG groups. PI3K immunohistochemical signals appear brown and the counterstaining background appears blue in (E) blank control, (F) vehicle control (G) PCOS and (H) DMBG groups. Higher magnification images of PI3K staining are presented for (I) blank control, (J) vehicle control, (K) PCOS and (L) DMBG groups. Scale bar, 100 µm. PI3K, phosphatidylinositol 3-kinase; PCOS, polycystic ovary syndrome; DMBG, dimethyldiguanide; GC, granulosa cell; Oo, oocyte.