. 2024 Jul 10;15(1):53.

doi: 10.1186/s13293-024-00630-2.

MicroRNA-21 modulates brown adipose tissue adipogenesis and thermogenesis in a mouse model of polycystic ovary syndrome

Samar Rezq^{1

2

3

4}, Alexandra M Huffman^{5

6

7}, Jelina Basnet^{8

5

6

7}, Amira E Alsemeh⁹, Jussara M do Carmo^{10

11}, Licy L Yanes Cardozo^{8

12

5

6

7}, Damian G Romero^{13

14

15

16}

Affiliations

¹ Department of Pharmacology and Toxicology, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA. srezq@umc.edu.
² Women's Health Research Center, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA. srezq@umc.edu.
³ Cardiovascular-Renal Research Center, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA. srezq@umc.edu.
⁴ Department of Cell and Molecular Biology, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA. srezq@umc.edu.
⁵ Women's Health Research Center, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA.
⁶ Cardiovascular-Renal Research Center, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA.
⁷ Department of Cell and Molecular Biology, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA.
⁸ Department of Pharmacology and Toxicology, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA.
⁹ Department of Anatomy, Histology, and Embryology, Faculty of Medicine, Zagazig University, Zagazig, Egypt.
¹⁰ Department of Physiology and Biophysics, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA.
¹¹ Cardiorenal and Metabolic Diseases Research Center, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA.
¹² Department of Medicine, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA.
¹³ Department of Pharmacology and Toxicology, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA. dromero@umc.edu.
¹⁴ Women's Health Research Center, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA. dromero@umc.edu.
¹⁵ Cardiovascular-Renal Research Center, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA. dromero@umc.edu.
¹⁶ Department of Cell and Molecular Biology, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA. dromero@umc.edu.

PMID: 38987854
PMCID: PMC11238487
DOI: 10.1186/s13293-024-00630-2

MicroRNA-21 modulates brown adipose tissue adipogenesis and thermogenesis in a mouse model of polycystic ovary syndrome

Samar Rezq et al. Biol Sex Differ. 2024.

. 2024 Jul 10;15(1):53.

doi: 10.1186/s13293-024-00630-2.

Authors

Affiliations

¹ Department of Pharmacology and Toxicology, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA. srezq@umc.edu.
² Women's Health Research Center, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA. srezq@umc.edu.
³ Cardiovascular-Renal Research Center, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA. srezq@umc.edu.
⁴ Department of Cell and Molecular Biology, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA. srezq@umc.edu.
⁵ Women's Health Research Center, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA.
⁶ Cardiovascular-Renal Research Center, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA.
⁷ Department of Cell and Molecular Biology, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA.
⁸ Department of Pharmacology and Toxicology, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA.
⁹ Department of Anatomy, Histology, and Embryology, Faculty of Medicine, Zagazig University, Zagazig, Egypt.
¹⁰ Department of Physiology and Biophysics, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA.
¹¹ Cardiorenal and Metabolic Diseases Research Center, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA.
¹² Department of Medicine, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA.
¹³ Department of Pharmacology and Toxicology, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA. dromero@umc.edu.
¹⁴ Women's Health Research Center, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA. dromero@umc.edu.
¹⁵ Cardiovascular-Renal Research Center, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA. dromero@umc.edu.
¹⁶ Department of Cell and Molecular Biology, University of Mississippi Medical Center, 2500 N. State Street, Jackson, MS, 39216, USA. dromero@umc.edu.

PMID: 38987854
PMCID: PMC11238487
DOI: 10.1186/s13293-024-00630-2

Abstract

Background: Polycystic ovary syndrome (PCOS), the most common endocrine disorder in premenopausal women, is associated with increased obesity, hyperandrogenism, and altered brown adipose tissue (BAT) thermogenesis. MicroRNAs play critical functions in brown adipocyte differentiation and maintenance. We aim to study the role of microRNA-21 (miR-21) in altered energy homeostasis and BAT thermogenesis in a PCOS mouse model of peripubertal androgen exposure.

Methods: Three-week-old miR-21 knockout (miR21KO) or wild-type (WT) female mice were treated with dihydrotestosterone (DHT) or vehicle for 90 days. Body composition was determined by EchoMRI. Energy expenditure (EE), oxygen consumption (VO2), carbon dioxide production (VCO2), and respiratory exchange ratio (RER) were measured by indirect calorimetry. Androgen receptor (AR), and markers of adipogenesis, de novo lipogenesis, angiogenesis, extracellular matrix remodeling, and thermogenesis were quantified by RT-qPCR and/or Western-blot.

Results: MiR-21 ablation attenuated DHT-mediated increase in body weight while having no effect on fat or BAT mass. MiR-21 ablation attenuated DHT-mediated BAT AR upregulation. MiR-21 ablation did not alter EE; however, miR21KO DHT-treated mice have reduced VO2, VCO2, and RER. MiR-21 ablation reversed DHT-mediated decrease in food intake and increase in sleep time. MiR-21 ablation decreased some adipogenesis (Adipoq, Pparγ, and Cebpβ) and extracellular matrix remodeling (Mmp-9 and Timp-1) markers expression in DHT-treated mice. MiR-21 ablation abolished DHT-mediated increases in thermogenesis markers Cpt1a and Cpt1b, while decreasing CIDE-A expression.

Conclusions: Our findings suggest that BAT miR-21 may play a role in regulating DHT-mediated thermogenic dysfunction in PCOS. Modulation of BAT miR-21 levels could be a novel therapeutic approach for the treatment of PCOS-associated metabolic derangements.

Keywords: Adipogenesis; Brown adipose tissue; Energy expenditure; Polycystic ovary syndrome; Thermogenesis.

Plain language summary

Polycystic ovary syndrome (PCOS) is a common hormone disorder in premenopausal women, often linked to obesity and abnormal brown fat tissue activity. Women with PCOS have elevated male hormones, which are responsible for many metabolic problems. Our study focuses on understanding the role of microRNA-21 (miR-21) in the energy balance and brown fat tissue activity in a PCOS mouse model. We studied female mice with and without miR-21, treating them with a male hormone. We measured body composition and energy expenditure. We also analyzed the levels of specific genes and proteins related to fat tissue and energy production. Our findings showed that mice lacking miR-21 had less weight gain in response to male hormones, without fat or brown fat tissue mass changes. They also had reduced energy production, changed eating habits, and altered expression of genes related to fat tissue and energy production. In conclusion, our study suggests that miR-21 in brown fat tissue may regulate the energy imbalance caused by male hormones in PCOS. Adjusting miR-21 levels in brown fat tissue could be a new way to address the metabolic issues associated with PCOS.

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Conflict of interest statement

LLYC is an Associate Editor of Biology of Sex Differences. DGR is an Editorial Board member of Biology of Sex Differences. No other conflicts of interest, financial or otherwise, are declared by the authors.

Figures

**Fig. 1**
Effect of miR-21 on DHT-mediated effects in body composition and BAT miR-21 and AR expression. WT or miR21KO mice were treated with dihydrotestosterone (DHT) or vehicle (Veh) for 90 days. A Body weight was determined by gravimetry, fat and lean masses were determined by EchoMRI. N = 6/group. B BAT mass was determined by gravimetry. N = 6/group. C BAT miR-21 was quantified by RT-qPCR, and expressed as arbitrary units (AU) standardized against SNORD110. N = 6/group. D BAT AR miRNA was quantified by RT-qPCR, and expressed as AU standardized to the geometric mean of four reference genes (GMHK). N = 4/group. E BAT AR protein expression was quantified by Western-blot normalized to GAPDH. N = 6 mice/group. Data are expressed as mean ± SEM. Data were analyzed by two-way ANOVA followed by Fisher’s LSD test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

**Fig. 2**
Effect of miR-21 on DHT-mediated changes in energy homeostasis, locomotion and sleep. WT or miR21KO mice were treated with dihydrotestosterone (DHT) or vehicle (Veh) for 90 days. A, B Energy expenditure (EE) was determined by indirect calorimetry (N = 6–7/group). C Food intake is expressed as Kcal and normalized by body weight (N = 6–8/group). D Sleep time, defined as the animal's immobility for more than 40 s, was measured in hr (N = 7–10/group). E, F Locomotor activity (E) was determined based on the number of infrared light beams breaks mounted in the cages in X, Y, and Z axes, and the total distance the animals moved in the cage (F) was measured in meters (N = 5–8/group). G, H Oxygen consumption (VO2) (G) and carbon dioxide production (VCO2) (H) was measured as the volume of gas produced/consumed/hr and normalized by body weight (N = 6–7/group). I, J The respiratory exchange ratio (RER) was calculated as the VCO2/VO2 ratio (N = 6–7/group). Data are expressed as mean ± SEM. Data were analyzed by two-way ANOVA followed by Fisher’s LSD test. *p < 0.05; **p < 0.01; ***p < 0.001

**Fig. 3**
DHT-mediated changes in BAT adipogenesis and de novo lipogenesis are modulated by miR-21. WT or miR21KO mice were treated with dihydrotestosterone (DHT) or vehicle (Veh) for 90 days. C/EBPα (Cebpa, A), C/EBPβ (Cebpb, C), Adiponectin (Adipoq, E), and PPARγ (pparg, G) mRNA was quantified by RT-qPCR and standardized to the geometric mean of four reference genes (HGMK) (N = 6/group). C/EBPα (B), C/EBPβ (D), Adiponectin (F), PPARγ (H), ACC (I), FAS (J), Fabp4 (K), and Perilipin 1 (L) protein was quantified by Western-blot and normalized to GAPDH (N = 4 mice/group). Data are expressed as mean ± SEM. Data were analyzed by two-way ANOVA followed by Fisher’s LSD test. *p < 0.05; **p < 0.01; ***p < 0.001. *ACC* acetyl-CoA carboxylase, *FAS* fatty acid synthase, *Fabp4* fatty acid binding protein 4

**Fig. 4**
MiR-21 modulates DHT-mediated changes in BAT adaptive thermogenesis targets. WT or miR21KO mice were treated with dihydrotestosterone (DHT) or vehicle (Veh) for 90 days. Cidea (A), Ppargc1a (B), and Prdm16 (C), Ucp-1 (E), Cpt1a (G), Cpt1b (I), Dio2 (K), Elovl3 (M), and Cox subunits (4i1, 4i2, 7a, and 8b) (O) mRNA was quantified by RT-qPCR and standardized to the geometric mean of four reference genes (HGMK) (N = 6/group). CIDE-A (B), UCP-1 (F), CPT1A (H), CPT1B (J), DIO2 (L), and COX4 (N) proteins were quantified by Western-blot and normalized to GAPDH (N = 4 mice /group). Data are expressed as mean ± SEM. Data were analyzed by two-way ANOVA followed by Fisher’s LSD test. *p < 0.05; **p < 0.01; ****p < 0.0001. *CPT1* carnitine palmitoyltransferase 1, *COX* cytochrome oxidase C, *DIO2* deiodinase 2

**Fig. 5**
Effect of DHT and miR-21 on BAT extracellular matrix remodeling and angiogenesis. WT or miR21KO mice were treated with dihydrotestosterone (DHT) or vehicle (Veh) for 90 days. BAT gene expression of Mmp-9 (A), Timp-1 (B), and Vegfa (C) was quantified by RT-qPCR and expressed as arbitrary unite (AU) standardized to the geometric mean of four reference genes (GMHK). Data are expressed as mean ± SEM (N = 6/group). Data were analyzed by two-way ANOVA followed by Fisher’s LSD test. *p < 0.05; **p < 0.01. *Mmp-9* matrix metalloproteinase-9, *Timp-1* tissue inhibitor of metalloproteinase-1, *Vegfa* vascular endothelial growth factor-A

**Fig. 6**
Effect of DHT and miR-21 on BAT histology. WT (A, C) or miR21KO (E, G) mice were treated with dihydrotestosterone (DHT) or vehicle (Veh) for 90 days. BAT sections were H&E stained and imaged. B, D, F, and H represent higher magnification of the regions (rectangles) in A, C, E, and G, respectively. The histopathological features are indicated as follows: Bifid arrow: small lipid droplets, arrowhead: connective tissue septa, circle: disorganized brown adipose tissues, short arrow: single large lipid droplet with irregular outline, and *: wide separation in certain tissue areas. Scale bar for A, C, E, and G = 200 μm, Scale bar for B, D, F, and G = 50 μm. BV normal blood vessel, *BV dilated and congested blood vessel, n peripheral signet nucleus, N central nucleus

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References

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MicroRNA-21 modulates brown adipose tissue adipogenesis and thermogenesis in a mouse model of polycystic ovary syndrome

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MicroRNA-21 modulates brown adipose tissue adipogenesis and thermogenesis in a mouse model of polycystic ovary syndrome

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Abstract

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Conflict of interest statement

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