MicroRNA-146b promotes adipogenesis by suppressing the SIRT1-FOXO1 cascade

Abstract

Sirtuin 1 (SIRT1) plays a critical role in the maintenance of metabolic homeostasis and promotes fat mobilization in white adipose tissue. However, regulation of SIRT1 during adipogenesis, particularly through microRNAs, remains unclear. We observed that miR-146b expression was markedly increased during adipogenesis in 3T3-L1 cells. Differentiation of 3T3-L1 was induced by overexpression of miR-146b. Conversely, inhibition of miR-146b decreased adipocyte differentiation. Bioinformatics-based studies suggested that SIRT1 is a target of miR-146b. Further analysis confirmed that SIRT1 was negatively regulated by miR-146b. We also observed that miR-146b bound directly to the 3'-untranslated region of SIRT1 and inhibited adipogenesis through SIRT1 downregulation. The miR-146b/SIRT1 axis mediates adipogenesis through increased acetylation of forkhead box O1 (FOXO1). Expression of miR-146b was increased and SIRT1 mRNA subsequently decreased in the adipose tissues of diet-induced and genetically obese mice. Furthermore, in vivo knockdown of miR-146b by a locked nucleic acid miR-146b antagomir significantly reduced body weight and fat volume in accordance with upregulation of SIRT1 and subsequent acetylation of FOXO1. Therefore, the miR-146b/SIRT1 pathway could be a potential target for obesity prevention and treatment.

Keywords: SIRT1; adipogenesis; fat mass; microRNA-146b; obesity.

Figure 1. miR-146b induces 3T3-L1 adipocyte differentiation

1. qRT-PCR analysis of miR-146b during 3T3-L1 differentiation (n = 4). Values are means ± SD. *p < 0.05 compared with day 0.

2. The effect of miR-146b activator (Ac) on miR-146b expression. Undifferentiated 3T3-L1 cells were transfected with miR-146 Ac or miR Ac control (CTL) and maintained in DMEM supplemented with 10% FBS for 8 d. Expression of miR-146b was calculated relative to MOCK (n = 3). Values are means ± SD. *p < 0.05 versus miR Ac CTL.

3. The effect of miR-146b Ac on intracellular lipid accumulation in undifferentiated 3T3-L1 cells. Lipid droplets were detected by Oil red O staining.

4. Intracellular lipid accumulation was quantified by measuring optical absorbance at 500 nm (n = 3). Values are means ± SD. *p < 0.05 versus miR Ac CTL.

5. Western blotting was performed for various adipocyte differentiation markers.

6. The effect of miR-146b inhibitor (In) on miR-146b expression. 3T3-L1 cells transfected with miR-146b or miR In CTL were differentiated according to a standard protocol. Expression of miR-146b was calculated relative to MOCK (n = 3). Values are means ± SD. *p < 0.05 versus miR In CTL.

7. The effect of miR-146b In on intracellular lipid accumulation in differentiated 3T3-L1 cells. Intracellular lipid accumulation was stained with Oil red O and quantitated by spectrophotometric measurement (n = 3). Values are means ± SD. *p < 0.05 versus miR In CTL.

8. Western blotting was performed for various adipocyte differentiation markers.

Figure 2. miR-146b negatively modulates SIRT1

1. The miR-146b sequence and its predicted binding site for the mouse and human SIRT1 mRNA sequences.

2. The effect of miR-146b Ac on the mRNA level of SIRT1 in undifferentiated 3T3-L1 cells. Expression of SIRT1 mRNA was calculated relative to MOCK (n = 3). Values are means ± SD. *p < 0.05 versus miR Ac CTL.

3. The effect of miR-146b Ac on expression of SIRT1 protein in undifferentiated 3T3-L1 cells.

4. The effect of miR-146b In on SIRT1 mRNA expression in differentiated 3T3-L1 cells. Expression of SIRT1 mRNA was calculated relative to MOCK (n = 3). Values are means ± SD. *p < 0.05 versus miR In CTL.

5. The effect of miR-146b In on expression of SIRT1 protein in differentiated 3T3-L1 cells.

6. 3T3-L1 cells were transfected with wild-type or mutant SIRT1 3′-UTR luciferase constructs and with miR-146b Ac, Ac CTL, miR-146b In or In CTL as indicated (n = 5). Values are means ± SD. *p < 0.05 versus miR Ac CTL. #p < 0.05 versus miR In CTL.

Figure 3. Downregulation of SIRT1 is required for the adipogenic effect of miR-146b

1. Preadipocytes transduced with Lenti-sh NC- or Lenti-sh SIRT1were transfected with miR-146b In or miR In CTL. Cells were stimulated to differentiate after 2 d. Differentiated cells were stained with Oil red O.

2. Intracellular lipid accumulation was stained with Oil red O and quantitated by spectrophotometric measurement (n = 3). Values are means ± SD. *p < 0.05 versus miR In CTL.

3. The effect of EX-527 on the anti-adipogenic activity of miR-146b In. Cells were pretreated with 10 µM EX-527 for 6 h and then switched to differentiation medium containing 10 µM EX-527. Cells were stained with Oil red O on day 8 of differentiation.

4. The effect of EX-527 on SIRT1 expression in the experiment described in (B). SIRT1 mRNA expression levels were measured by qRT-PCR (n = 3). Values are means ± SD. *p < 0.05 versus miR In CTL.

5. After 2 d of transfection with miR-146b Ac or miR-Ac CTL, 3T3-L1 cells were maintained in growth medium for 8 d in the presence of 5 µM SA-3. Cells were then stained with Oil red O.

6. The effect of SA-3 on SIRT1 protein expression in the experiment described in (D). Western blot analysis was performed for SIRT1 in total protein extracts.

Figure 4. miR-146b/SIRT1 pathway controls the deacetylation of FOXO1

1. The effect of miR-146b Ac on acetylation of FOXO1 in undifferentiated 3T3-L1 cells. Total and acetylated FOXO1 (Ac-FOXO1) was detected by Western blotting.

2. Ac-FOXO1/FOXO1 ratio was based on densitometric quantifications of Ac-FOXO1 and total FOXO1 levels on Western blots (n = 3). *p < 0.05 versus miR Ac CTL.

3. The effect of miR-146b In on acetylation of FOXO1 in differentiated 3T3-L1 cells. Total and Ac-FOXO1 was detected by Western blotting.

4. Ac-FOXO1/FOXO1 ratio was based on densitometric quantifications of Ac-FOXO1 and total FOXO1 levels on Western blots (n = 3). *p < 0.05 versus miR In CTL.

Figure 5. Silencing of miR-146b ameliorates obesity through upregulation of SIRT1

1. Knockdown of miR-146b by LNA-miR-146b antagomir (LNA-miR-146b) and the subsequent increase in SIRT1 expression were confirmed through qRT-PCR analysis in the epididymal fat tissue of obese C57BL/6J mice. Mice were intraperitoneally injected with 25 mg/kg/d LNA-miR-146b or LNA-scramble for 3 consecutive days and sacrificed 72 h after the last injection (n = 5). Values are means ± SEM. *p < 0.05 versus LNA-scramble.

2. The effect of miR-146b knockdown by LNA-miR-146b on FOXO1 acetylation. Total and Ac-FOXO1 was detected by Western blotting.

3. Knockdown of miR-146b by LNA-miR-146b induced weight loss in mice. Body weight was measured 72 h after the last injection (n = 5). Values are means ± SEM. ns, not significant; *p < 0.05, **p < 0.01.

4. Knockdown of miR-146b by LNA-miR-146b reduced visceral fat volume. CT images were acquired from anaesthetized mice and analysed to calculate visceral fat mass (n = 5). Values are means ± SEM. ns, not significant; *p < 0.05, **p < 0.01.

5. Knockdown of miR-146b by LNA-miR-146b induced hypotrophy of white adipose tissues. Perirenal fat fads were stained with H&E (left), and the mean sizes of adipocytes were calculated (right) (n = 5). Scale bar = 100 µm. Values are means ± SEM. *p < 0.05 versus LNA-scramble.

6. Western blot analysis on adipogenesis-related markers of white adipose tissue from LNA-146b-injected mice. Expressions of C/EBPα, PPARγ, fatty acid synthase and AP2 of perirenal fat tissues were measured. β-actin was used as a loading control.

Figure 6. Schematic representation of the pro-adipogenic effect of miR-146b

Adipogenic stimuli induce expression of miR-146b. Increased miR-146b binds directly to SIRT1 and downregulates SIRT1 expression. SIRT1 downregulation reduces deacetylation of FOXO1. As a result, FOXO1-mediated inhibition of PPARγ transcription is diminished and adipocyte differentiation is stimulated.