A novel role for the Wnt inhibitor APCDD1 in adipocyte differentiation: Implications for diet-induced obesity

Abstract

Impaired adipogenic differentiation during diet-induced obesity (DIO) promotes adipocyte hypertrophy and inflammation, thereby contributing to metabolic disease. Adenomatosis polyposis coli down-regulated 1 (APCDD1) has recently been identified as an inhibitor of Wnt signaling, a key regulator of adipogenic differentiation. Here we report a novel role for APCDD1 in adipogenic differentiation via repression of Wnt signaling and an epigenetic linkage between miR-130 and APCDD1 in DIO. APCDD1 expression was significantly up-regulated in mature adipocytes compared with undifferentiated preadipocytes in both human and mouse subcutaneous adipose tissues. siRNA-based silencing of APCDD1 in 3T3-L1 preadipocytes markedly increased the expression of Wnt signaling proteins (Wnt3a, Wnt5a, Wnt10b, LRP5, and β-catenin) and inhibited the expression of adipocyte differentiation markers (CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ)) and lipid droplet accumulation, whereas adenovirus-mediated overexpression of APCDD1 enhanced adipogenic differentiation. Notably, DIO mice exhibited reduced APCDD1 expression and increased Wnt expression in both subcutaneous and visceral adipose tissues and impaired adipogenic differentiation in vitro Mechanistically, we found that miR-130, whose expression is up-regulated in adipose tissues of DIO mice, could directly target the 3'-untranslated region of the APCDD1 gene. Furthermore, transfection of an miR-130 inhibitor in preadipocytes enhanced, whereas an miR-130 mimic blunted, adipogenic differentiation, suggesting that miR-130 contributes to impaired adipogenic differentiation during DIO by repressing APCDD1 expression. Finally, human subcutaneous adipose tissues isolated from obese individuals exhibited reduced expression of APCDD1, C/EBPα, and PPARγ compared with those from non-obese subjects. Taken together, these novel findings suggest that APCDD1 positively regulates adipogenic differentiation and that its down-regulation by miR-130 during DIO may contribute to impaired adipogenic differentiation and obesity-related metabolic disease.

Keywords: APCDD1; Wnt signaling; adipocyte; adipose tissue; differentiation; miR-130; obesity.

Figure 1.

Wnt and APCDD1 expression in relation to adipogenic differentiation. A, expression of Wnt1, Wnt3a, and Wnt10b mRNAs was down-regulated during adipogenic differentiation (6 days) of human subcutaneous preadipocytes (n = 3). *, p < 0.01; **, p < 0.05 versus control (Day 0). B, the APCDD1 mRNA level was up-regulated in mature human adipocytes (AD) compared with PAs within the SV fraction (n = 3). *, p < 0.01 versus control (SV). C, APCDD1 mRNA expression was increased during in vitro adipogenic differentiation (12 days) of primary cultured human preadipocytes (n = 3). *, p < 0.01 versus control (Day 0). D, time course expression of APCDD1 mRNA during adipogenic differentiation of 3T3-L1 preadipocytes (n = 3). *, p < 0.05 versus control (Day 0). E and F, APCDD1 protein expression was increased during in vitro adipogenic differentiation (12 days) of 3T3-L1 preadipocytes and primary cultured mouse preadipocytes (n = 3). *, p < 0.05 versus control (Day 0). Levels of mRNA and protein expressions were determined by qPCR and Western blotting, respectively.

Figure 2.

APCDD1 gene silencing suppresses adipogenic differentiation in conjunction with up-regulated expression of Wnt signaling proteins in 3T3-L1 preadipocytes. A, adipogenic differentiation was impaired in 3T3-L1 preadipocytes transfected with APCDD1 siRNA compared with the scrambled control, as indicated by reduced cytoplasmic lipid droplet accumulation (Oil red O). ****, p < 0.0001 versus control (Scramble). B and C, transfection of 3T3-L1 preadipocytes with APCDD1 siRNA increased expression of Wnt signaling proteins (Wnt3a, Wnt5a, Wnt10b, LRP5, and β-catenin) and decreased expression of adipogenic markers (C/EBPα and PPARγ) (n = 3). β-catenin, C/EBPα, and PPARγ expression levels were examined in the nuclear fraction. *, p < 0.01; **, p < 0.05 versus control. Protein expressions were determined by Western blotting and densitometry analysis. Scr, scrambled.

Figure 3.

APCDD1 overexpression accelerates adipogenic differentiation and down-regulates the expression of Wnt signaling proteins in 3T3-L1 preadipocytes. A, adipogenic differentiation, as indicated by lipid droplet accumulation (Oil red O), was increased in 3T3-L1 preadipocytes transfected with adenoviral APCDD1 compared with the control. *, p < 0.01 versus control (Adeno-GFP). B, mRNA expression of adipogenic markers (adiponectin and FABP4) was up-regulated in adenoviral APCDD1-transduced 3T3-L1 preadipocytes during differentiation (7 days) (n = 4). *, p < 0.01 versus control (PA); **, p < 0.05 versus GFP. AD, adipocyte. C and D, transduction of 3T3-L1 preadipocytes with adenoviral APCDD1 down-regulated Wnt-associated protein expression (Wnt3a, Wnt5a, Wnt10b, LRP5, and β-catenin) and up-regulated adipogenic marker (C/EBPα and PPARγ) protein expression during differentiation (n = 3). *, p < 0.01; **, p < 0.05 versus control (CTL). Levels of mRNA and protein expressions were determined by qPCR and Western blotting, respectively. Representative blots are shown in C and densitometry in D.

Figure 4.

Reduced APCDD1 expression is associated with up-regulated Wnt3a and blunted adipogenic differentiation in DIO mice. A, 18 weeks of HFD feeding, compared with CD feeding, significantly increased body weight in mice (n = 3). B—D, APCDD1 mRNA (B) and protein (C) levels were down-regulated, whereas Wnt3a was up-regulated, in extracts of SQ (C) and visceral (D) adipose tissues isolated from HFD-fed DIO mice compared with lean controls (n = 3). Relative mRNA and protein expression were quantified by qPCR and Western blotting, respectively. *, p < 0.01; **, p < 0.05 versus control (CD). E, adipogenic differentiation (7 days) was impaired in preadipocytes isolated from subcutaneous adipose tissues of DIO mice. Light microscopy of neutral cytoplasmic lipid droplet accumulation was assessed by Oil red O staining.

Figure 5.

HFD-induced miR-130 targets APCDD1 and inhibits adipogenic differentiation. A, computational analysis predicted that miR-130a-3p and miR-130b-3p can directly bind the 3′ UTR of APCDD1. B, miR-130a-3p and miR103b-3p levels were down-regulated during adipogenic differentiation in 3T3-L1 preadipocytes. *, p < 0.05 versus control (Day 0). C, APCDD1 protein expression was increased by transfection of miR-130 inhibitor in 3T3-L1 preadipocytes (n = 3). *, p < 0.05 versus control (CTR). SCR, scramble. D, miR-130a-3p and miR-130b-3p levels were increased in the SQ and visceral adipose depots of HFD-fed DIO mice (n = 5). *, p < 0.01; **, p < 0.05 versus control (CD). E, miR-130a-3p expression was significantly reduced or increased by transfection of miR-130a-3p inhibitor or mimic, respectively, in 3T3-L1 preadipocytes (n = 3). *, p < 0.01 versus control. F, adipogenic differentiation of 3T3-L1 preadipocytes was enhanced by transfection of miR-130a-3p inhibitor and reduced by transfection of miR-130a-3p mimic. *, p < 0.0001 versus control. Light microscopy of neutral cytoplasmic lipid droplets accumulation was evaluated by Oil red O staining and optical density measurement by spectrophotometer.

Figure 6.

Down-regulated expression of APCDD1 in adipose tissues from obese humans is associated with reduced adipogenic gene expression. A and B, protein expression of APCDD1, in conjunction with C/EBPα, PPARγ, was down-regulated, whereas Wnt3a levels tended to be up-regulated (p = 0.06), in subcutaneous adipose tissues isolated from obese (BMI > 30) compared with non-obese subjects (BMI < 30). *, p < 0.05 versus non-obese control. Protein expressions were analyzed by Western blotting (A, representative data) and densitometry (B, n = 8).

Figure 7.

Schematic depicting the potential mechanisms of APCDD1-mediated adipogenic differentiation in the lean state versus DIO. Under normal physiological conditions, APCDD1 inhibits the expression of Wnt signaling proteins, leading to the induction of key adipogenic transcription factors (C/EBPα and PPARγ) and adipogenic differentiation. In DIO, HFD-induced miR-130 in adipose tissues blocks APCDD1 gene expression, thereby augmenting Wnt signaling and repressing adipogenic differentiation.