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. 2021 Nov 29:12:747944.
doi: 10.3389/fendo.2021.747944. eCollection 2021.

Brown Adipose Transplantation Improves Polycystic Ovary Syndrome-Involved Metabolome Remodeling

Lihua Yao  1   2 , Qin Wang  1   2 , Runjie Zhang  1   2 , Xingyun Wang  1   2 , Yiwen Liu  1   2 , Fangfang Di  1   2 , Liwen Song  1   2 , Siliang Xu  3
Affiliations

Brown Adipose Transplantation Improves Polycystic Ovary Syndrome-Involved Metabolome Remodeling

Lihua Yao et al. Front Endocrinol (Lausanne). .

Abstract

Polycystic ovary syndrome (PCOS) is a complex reproductive, endocrine, and metabolic disorder in reproductive-age women. In order to explore the active metabolites of brown adipose tissue (BAT) transplantation in improving the reproductive and metabolic phenotypes in a PCOS rat model, the metabolites in the recipient's BAT were explored using the liquid chromatography-mass spectrometry technique. In total, 9 upregulated and 13 downregulated metabolites were identified. They were roughly categorized into 12 distinct classes, mainly including glycerophosphoinositols, glycerophosphocholines, and sphingolipids. Ingenuity pathway analysis predicted that these differentially metabolites mainly target the PI3K/AKT, MAPK, and Wnt signaling pathways, which are closely associated with PCOS. Furthermore, one of these differential metabolites, sphingosine belonging to sphingolipids, was randomly selected for further experiments on a human granulosa-like tumor cell line (KGN). It significantly accelerated the apoptosis of KGN cells induced by dihydrotestosterone. Based on these findings, we speculated that metabolome changes are an important process for BAT transplantation in improving PCOS. It might be a novel therapeutic target for PCOS treatment.

Keywords: LC-MS; PCOS; brown adipose tissue; metabolites; sphingosine.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
BAT transplantation in a PCOS rat model. (A) Scheme of the group assignments and timeline of the experiment process (n = 24). Female SD rats were treated with DHEA for 21 days to construct the PCOS model. BAT transplantation or sham operation was performed on day 21. Donor rats, within 14 days after birth, were operated on to take BAT and then BAT was transplanted to PCOS model rats. The estrous cycles were checked daily for the following 10 days. Reproductive and metabolism phenotype detection was done on day 42. (B) Serum concentrations of FSH, LH, and AMH, as well as the LH/FSH ratio (5 control rats and 10 DHEA rats). (C). Disordered estrous cycles were observed in PCOS rats. (D) H&E staining of the ovarian tissues from the Ctrl and DHEA+Sham groups (scale bar, 1 mm). Ovarian histology revealed that cystic follicles (arrow) and a few corpora lutea (asterisk) appeared in the DHEA group compared with the Ctrl group. (E) UCP1 was identified by immunohistochemistry in the donor BAT (scale bar, 100 µm). PCOS, polycystic ovary syndrome; BAT, brown adipose tissue; SD, Sprague–Dawley; DHEA, dehydroepiandrosterone; FSH, follicle-stimulating hormone; H&E, hematoxylin and eosin; LH, luteinizing hormone; AMH, anti-Müllerian hormone; UCP1, uncoupling protein 1; D, diestrus; E, estrus; M, metestrus; P, proestrus. Data were analyzed using unpaired Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 2
Figure 2
Brown adipose tissue (BAT) transplantation improved the reproductive phenotype of polycystic ovary syndrome (PCOS) model rats. (A) Disordered estrous cycles were observed in the DHEA+Sham group, while BAT transplantation rescued the abnormal estrous cycles. (B) Representative results of ovarian H&E staining of the control (Ctrl) group, DHEA+Sham group, and DHEA+BAT group. Ovarian histology revealed that cystic follicles (arrow) and a few corpora lutea (asterisk) appeared in the DHEA+Sham group compared with the Ctrl group, while BAT transplantation reversed the phenotype caused by dehydroepiandrosterone (DHEA). H&E, hematoxylin–eosin; D, diestrus; E, estrus; M, metestrus; P, proestrus; UCP1, uncoupling protein 1.
Figure 3
Figure 3
Brown adipose tissue (BAT) transplantation partially corrected the metabolic abnormality of polycystic ovary syndrome (PCOS) model rats. (A) Comparison of the mean weight among the control (Ctrl) group, the DHEA/DHEA+Sham group, and the DHEA+BAT group. (B, C) Results of the GTT (B) and ITT (C) showed that insulin resistance was rescued by BAT transplantation in PCOS model rats. (D) UCP1 expression was reduced in the DHEA+Sham group and was enhanced in the DHEA+BAT group. GTT, glucose tolerance test; ITT, insulin tolerance test; UCP1, uncoupling protein 1. Data were analyzed using one-way ANOVA with post-hoc Scheffe test. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., no significance.
Figure 4
Figure 4
Score plot of the PLS-DA model. (A) Score plot of the PLS-DA model in the positive and the negative mode. (B) Permutation test of the PLS-DA model in the positive and the negative mode. Green dots indicate R 2 and blue dots indicate Q 2. PLS-DA, partial least squares discriminant analysis.
Figure 5
Figure 5
Differential analysis of the metabolites in brown adipose tissue (BAT) from the DHEA+Sham and DHEA+BAT groups. (A) Heatmap of the 22 metabolites that were differentially expressed between the DHEA+Sham (n = 7) and DHEA+BAT (n = 7) groups. Blue to red equates to an increase in metabolite expression. (B) Volcano plot of all metabolites expressed in the DHEA+Sham and DHEA+BAT groups. (C) KEGG pathway analysis of the differentially expressed metabolites. (D) All matched pathways are displayed as circles analyzed with MetaboAnalyst 3.0. Potential target pathways were selected either by impact values from pathway topology analysis or by negative log p-values from pathway enrichment analysis. The size of the bubble represents the number of metabolites enriched. KEGG, Kyoto Encyclopedia of Genes and Genomes.
Figure 6
Figure 6
Classification of the differential metabolites and IPA. (A) Classification of the differential metabolites was roughly categorized into 12 distinct classes. (B) IPA of the metabolites related to biological network, pathways, and functions. The differential metabolites were closely associated with the PI3K/AKT, MAPK, and Wnt signaling pathways. IPA, ingenuity pathway analysis.
Figure 7
Figure 7
Selected differentially expressed metabolites. (A–C) The glycerophosphoinositols and sphingolipids were decreased, while glycerophosphocholines were increased in the DHEA+BAT group. (D, E) Aldehydes and amino acids were reduced in the DHEA+BAT group compared with that in the DHEA+Sham group. (F) Peptides were increased in the DHEA+BAT group. Data were analyzed using unpaired Student’s t-test. *p < 0.05, **p < 0.01.
Figure 8
Figure 8
Analysis of the cell apoptosis of KGN cells transfected with sphingosine. The early and late apoptosis rates of KGN cells were compared using PI and FITC. Sphingosine increased the apoptosis of DHT-treated KGN cells. The results are triplicates. DHT, dihydrotestosterone; PI, propidium iodide; FITC, fluorescein isothiocyanate. Data were analyzed with unpaired Student’s t-test. **p < 0.01.
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