5-Aza-2'-Deoxycytidine Regulates White Adipocyte Browning by Modulating miRNA-133a/Prdm16

Abstract

The conversion of white adipocytes into brown adipocytes improves their thermogenesis and promotes energy consumption. Epigenetic modifications affect related genes and interfere with energy metabolism, and these are the basis of new ideas for obesity treatment. Neonatal mice show high levels of DNA hypermethylation in white adipose tissue early in life and low levels in brown adipose tissue. Thus, we considered that the regulation of DNA methylation may play a role in the conversion of white adipose to brown. We observed growth indicators, lipid droplets of adipocytes, brown fat specific protein, and miRNA-133a after treatment with 5-Aza-2'-deoxycytidine. The expression of Prdm16 and Ucp-1 in adipocytes was detected after inhibiting miRNA-133a. The results showed a decrease in total lipid droplet formation and an increased expression of the brown fat specific proteins Prdm16 and Ucp-1. This study indicated that 5-Aza-2'-deoxycytidine promotes white adipocyte browning following DNA demethylation, possibly via the modulation of miR-133a and Prdm16.

Keywords: 5-Aza-dC; Prdm16; Ucp-1; brown adipocytes; white adipocytes.

Figure 1

Graphic of the animal model design.

Figure 2

The effect of 5-Aza-dC on the growth characteristics of mice. (A) Identification of 5-methyl cytosine levels after 5-Aza-dC treatment, presented as the mean ± SD (n = 5). (B) Body weights in the NC, OB, OB-R, and 5-Aza-dC groups from weeks 1 to 12 are presented as the mean ± SD (n = 5). (C) Body lengths of mice in the four groups from weeks 2 to 12 are presented as the mean ± SD (n = 5). (D) Lee indices in the four groups at week 12 are presented as the mean ± SD (n = 5). (E) Four mice from the same litter; the first shows the general appearance of a normal mouse, the second shows an obese mouse, the third shows an obesity-resistant mouse, and the fourth shows a 5-aza-2′-deoxycytidine-induced mouse. (F) Body temperatures in the three groups from weeks 4 to 12 are shown as the mean ± SD (n = 5). (G) Levels of the four groups’ leptin levels in mouse serum are presented as the mean ± SD (n = 5). * p < 0.05; ** p < 0.01; *** p < 0.001 vs. the NC group; # p < 0.05; ## p < 0.01; ### p < 0.001, compared with each other. NC, normal control; OB, MSG-induced obese mice; OB-R, MSG-induced obesity-resistant mice; 5-Aza-dC, 5-aza-2′-deoxycytidine-induced mice.

Figure 3

Detection of the subcutaneous adipose tissue in mice after 5-Aza-dC treatment. (A) Body fat percentages of the four groups are presented as the mean ± SD (n = 5); (B) Fat weights of mice in the four groups are presented as the mean ± SD (n = 5); (C) WAT distribution in mice in the four groups is presented as the mean ± SD (n = 5); (D) The proportion of brown fat weight to total fat weight in the four groups, presented as the mean ± SD (n = 5). (E) Pictures of subcutaneous white adipose tissue of the four groups; (F) H&E staining in the inguinal subcutaneous WAT of mice induced by MSG (scale bar = 110 μm). * p < 0.05; ** p < 0.01; *** p < 0.001 vs. the NC group; ## p < 0.01; ### p < 0.001 vs. each other group. NC, normal control; OB, MSG-induced obese mice; OB-R, MSG-induced obesity-resistant mice; 5-Aza-dC, 5-aza-2′-deoxycytidine-induced DNA demethylation mice; WAT, white adipose tissue; BAT, brown adipose tissue.

Figure 4

Prdm16, Ucp-1, and miR-133a expression in subcutaneous WAT. (A–P) Increased immunoreactivity of Prdm16 (green) and Ucp1 (red) after DNA demethylation (n = 3/group; blue, DAPI). (Q,R) Expression of Prdm16 and Ucp1 in subcutaneous WAT using Western blotting. (S,T) Quantified changes in Prdm16 and Ucp1 after DNA demethylation (n = 3/group). (U,V) Normalized intensity of Prdm16 and Ucp1 relative to GAPDH, presented as the mean ± SD (n= 3/group). (W) The expression levels of miR-133a in the subcutaneous WAT of the four groups (n= 3/group). *** p < 0.001; ** p < 0.01; * p < 0.05, compared with controls; # p < 0.05; ## p < 0.001; ### p < 0.001, compared with each other. NC, normal control; OB, MSG-induced obese mice; OB-R, MSG-induced obesity-resistant mice; 5-Aza-dC, 5-aza-2′-deoxycytidine-induced DNA demethylation mice; Prdm16, PR domain containing 16; Ucp-1, uncoupling protein-1; WAT, white adipose tissue.

Figure 5

Detection of Prdm16, Ucp-1, and miR-133a expression in adipocytes (A–D). Primary adipocyte culture for the 0–12th day (A–D, Bar = 100 µm). (E) The growth of cells on the 12th day after DNA demethylation treatment; (F) The growth of cells on the 12th day (E,F, Bar = 100 µm); (G) Oil red staining of cells on the 12th day after DNA demethylation treatment; (H) Oil red staining of cells on the 12th day (G,H, Bar = 50 µm). (I,J) Expression of Prdm16 and Ucp1 in adipocytes using Western blotting. (K,L) Normalized intensity of Prdm16 and Ucp1 relative to GAPDH is presented as the mean ± SD (n= 3). (M) The expression levels of miR-133a in fully differentiated adipocytes treated with 5-aza. *** p < 0.001, ** p < 0.01, compared with controls. NC, normal control; 5-Aza-dC, 5-aza-2′-deoxycytidine-induced adipocytes.

Figure 6

Immunoreactivity of Prdm16/Ucp1 proteins after the inhibition of miR-133a expression. (A) The expression levels of miR-133a in fully differentiated adipocytes treated with 5-aza and transfected with or without miR-133a inhibitor. (B–M) Increased immunoreactivity of Prdm16 (green) and Ucp1 (red) in fully differentiated adipocytes treated with 5-aza and transfected with or without miR-133a inhibitor (n = 3/group; blue, DAPI). (N,O) Expression of Prdm16 and Ucp1 in fully differentiated adipocytes treated with 5-aza and transfected with or without miR-133a inhibitor using Western blotting. (P,Q) Quantified changes in Prdm16 and Ucp1 in fully differentiated adipocytes treated with 5-aza and transfected with or without miR-133a inhibitor. (R,S) Normalized intensity of Prdm16 and Ucp1 relative to GAPDH, presented as the mean ± SD (n= 3). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control group; # p < 0.05, ## p < 0.01 compared with other groups; bar = 130 μm. Prdm16, PR domain containing 16; Ucp-1, uncoupling protein-1; miR-133a, microRNA-133a; 5-aza-dC, 5-aza-2′-deoxycytidine.

Figure 7

Graphic of 5-Aza-dC-mediated browning of white adipocytes. 5-Aza-dC suppresses the expression of miR-133a, which is a direct inhibitor of Prdm16. Thus, the 5-Aza-dC-mediated suppression of miR-133a upregulates the expression of Prdm16 and promotes the browning of white fat, increasing Ucp-1 expression and thermogenic activity, which contributes to resistance to weight gain. miR-133a, microRNA-133a; Prdm16, PR domain containing 16; Ucp-1, uncoupling protein 1.