Increased IGFBP2 Levels by Placenta-Derived Mesenchymal Stem Cells Enhance Glucose Metabolism in a TAA-Injured Rat Model via AMPK Signaling Pathway

Abstract

The insulin resistance caused by impaired glucose metabolism induces ovarian dysfunction due to the central importance of glucose as a source of energy. However, the research on glucose metabolism in the ovaries is still lacking. The objectives of this study were to analyze the effect of PD-MSCs on glucose metabolism through IGFBP2-AMPK signaling and to investigate the correlation between glucose metabolism and ovarian function. Thioacetamide (TAA) was used to construct a rat injury model. PD-MSCs were transplanted into the tail vein (2 × 10⁶) 8 weeks after the experiment started. The expression of the IGFBP2 gene and glucose metabolism factors (e.g., AMPK, GLUT4) was significantly increased in the PD-MSC group compared to the nontransplantation (NTx) group (* p < 0.05). The levels of follicular development markers and the sex hormones AMH, FSH, and E2 were also higher than those in the TAA group. Using ex vivo cocultivation, the mRNA and protein expression of IGFBP2, AMPK, and GLUT4 were significantly increased in the cocultivation with the PD-MSCs group and the recombinant protein-treated group (* p < 0.05). These findings suggest that the increased IGFBP2 levels by PD-MSCs play an important role in glucose metabolism and ovarian function through the IGFBP2-AMPK signaling pathway.

Keywords: AMPK; IGFBP2; mitochondria; ovarian dysfunction; placenta-derived mesenchymal stem cells.

Figure 1

Thioacetamide-induced ovarian dysfunction and insulin resistance and impaired glucose metabolism. (A) Glucose, (B) insulin, (C) albumin, (D) total bilirubin, (E) ALT, and (F) AST levels were analyzed in serum by ELISA. (G,H) The HOMA-IR was performed according to the following equation: glucose (mg/dL) × insulin (mU/mL)/405; HOMA-IS was calculated according to the following equation: 1/HOMA-IR. (I) A morphological analysis in ovarian tissue of TAA- injury rats after sacrifice. (J) A morphological analysis in liver tissue of TAA-injury rats after sacrifice. Scale bar: 5 mm. (K) The ratio of ovarian weight to body weight analyzed after sacrifice. (L) The apoptotic cells of follicles were stained with a TUNEL assay kit. Scale bar: 100 μm; magnification: 20X. (M) The TUNEL intensity was quantified by 3D Histech. (N) The localization of LC3b protein expression in ovarian follicles analyzed using immunofluorescence (white arrow). Scale bar: 50 μm; magnification: 20X. (O) The localization of LC3b was quantified using ImageJ. (P) The protein expression was analyzed using western blotting.And its densitometry quantification showed the upregulation of (Q) LC3b and (R) BECN1 and the downregulation of (S) mTOR after PD-MSC transplantation. GC: granulosa cell; TC: theca cell. The data represent the mean ± S.D. Statistical significance was determined using a one-way ANOVA. Note: * p < 0.05, Nor vs. NTx; * p < 0.05, NTx vs. Tx.

Figure 2

Glucose metabolism and insulin pathway signaling activated by PD-MSCs through the AMPK signaling pathway. Glucose metabolism and insulin pathway signaling are activated by PD-MSCs through the AMPK signaling pathway. (A) The localization of GLUT4 in mature follicles (e.g., antral follicles) of ovarian tissues was analyzed via immunohistochemistry staining. Scale bar: 100 μm; magnification: 40X. (B) The relative DAB intensity was analyzed by 3D Histech. (C) The localization of IGFBP2 in ovarian tissue was analyzed using immunofluorescence. Scale bar: 50 μm; magnification: 20X. (D) IGFBP2 gene expression was analyzed using the ImageJ program. The mRNA expression of IRS1 (E) and IR (F) was analyzed using qRT-PCR. Western blotting (G) and protein expression of AMPK (H), SIRT1 (I), GLUT4 (J), IGFBP2 (K), FOXO3a (L), PI3K (M), AKT (N), and IR (O) were quantified. (P) The concentration of ATP production was determined in serum via ELISA. (Q) The ratio of mitochondrial DNA in the gDNA of ovarian tissues was analyzed via qRT-PCR. GC: granulosa cell; TC: theca cell. The data represent the mean ± S.D. Statistical significance was determined using a one-way ANOVA. Note: * p < 0.05, Nor vs. NTx; * p < 0.05, NTx vs. Tx.

Figure 3

Effect of PD-MSCs on ovarian function in the ovaries of the TAA-injured rats. (A) The levels of AMH, (B) E2, (C) FSH, and (D) testosterone in individual serum samples were analyzed via ELISA. (E) A histological analysis of follicular development was performed using H&E staining. Scale bar: 2 mm; magnification: 1.4×. (F) The follicular count according to follicular development was analyzed via serial sectioned ovarian tissues. (G–K) The protein expression of Nanos3, Nobox, BMP15, and EGFR related to folliculogenesis was analyzed using Western blotting. The data represent the mean ± S.D. Statistical significance was determined using a one-way ANOVA. Note: * p < 0.05, Nor vs. NTx; * p < 0.05, NTx vs. Tx.

Figure 4

An increase in IGFBP2 cytokines secreted by PD-MSCs activated glucose metabolism in the TAA-injured rat ovary model. (A) Scheme of the ex vivo experiments. Ovaries were harvested and treated with PD-MSCs, recombinant IGFBP2, and PPP (IGF-1 inhibitor). (B–G) The mRNA expression of AMPK, SIRT1, GLUT4, IR, IGF-1, and IGFBP2 was analyzed using qRT-PCR. (H–J) The protein expression of AMPK, GLUT4, and IGFBP2 was analyzed via Western blotting. The data represent the mean ± S.D. Statistical significance was determined by using a one-way ANOVA. Note: * p < 0.05, Nor vs. TAA treatment; # p < 0.05, NTx vs. cocultivation with PD-MSCs; recombinant IGFBP2, inhibitor, $ p < 0.05 cocultivation with PD-MSCs vs. recombinant IGFBP2; ## p < 0.05 inhibitor vs. inhibitor + PD-MSCs.

Figure 5

The activation of glucose metabolism markers was enhanced by the AMPK signaling pathway in granulosa cells (in vitro). (A) Scheme of the in vitro experiment. KGN cells and primary theca cells were seeded in 6-well plates and treated with 70 mM TAA and PD-MSCs. (B–E) In theca cells, the mRNA expression of IGFBP2, IGF-1, AMPK, SIRT1, and GLUT4 was analyzed via qRT-PCR. (F–J) In granulosa cells, the mRNA expression of IGFBP2, IGF-1, AMPK, and SIRT1 was analyzed via qRT-PCR. (K) The protein expression of AMPK and GLUT4 was analyzed using Western blotting. (L,M) The Western blot bands of AMPK and GLUT4 gene were quantified using ImageJ. The data represent the mean ± S.D. Statistical significance was determined by using a one-way ANOVA. Note: * p < 0.05, theca cell/granulosa cell vs. NTx; * p < 0.05, NTx vs. Tx.