Investigating the crosstalk between ABCC4 and ABCC5 in 3T3-L1 adipocyte differentiation

Abstract

Introduction: The plasma membrane-bound protein, multi-drug resistance-associated protein 4 (MRP4/ABCC4), has gained attention for its pivotal role in facilitating the efflux of a wide range of endogenous and xenobiotic molecules. Its significance in adipogenesis and fatty acid metabolism has been brought to light by recent studies. Notably, research on ABCC4 knockout (ABCC4 ^-/- ) mice has established a link between the absence of ABCC4 and the development of obesity and diabetes. Nevertheless, the specific contribution of ABCC4 within adipose tissue remains largely unexplored.

Methods: To address this gap, we conducted a study to elucidate the role of the ABCC4 transporter in mature adipocytes, using siRNA constructs to silence its gene function.

Results: The successful knockdown of ABCC4 significantly altered lipid status and adipogenic gene expression in mature 3T3-L1 adipocytes. Intriguingly, this knockdown also altered the gene expression patterns of other ABCC transporter family members in 3T3-L1 cells. The downregulation of ABCC5 expression was particularly noteworthy, suggesting potential crosstalk between ABCC transporters in mature adipocytes. Additionally, knocking down ABCC5 resulted in significantly higher adipogenic and lipogenic gene expression levels. Oil Red O staining confirmed increased lipid accumulation following the knockdown of ABCC4 and ABCC5. Surprisingly, the simultaneous knockdown of both transporters did not show a cumulative effect on adipogenesis, rather it led to higher levels of intracellular cAMP and extracellular prostaglandin metabolite, both of which are essential signaling molecules in adipogenesis.

Conclusion: These results highlight the complex interplay between ABCC4 and ABCC5 transporters in adipocyte function and suggest their individual contributions toward obesity and related disorders.

Keywords: 3T3-L1 cells; ABCC transporter; adipogenesis; cAMP; lipids; siRNA.

FIGURE 1

3T3-L1 cell differentiation and expression of ATP-binding cassette transporters. (A) ABCC4 gene expression levels in 3T3-L1 cells at indicated phases of differentiation. Gene expression levels were normalized with GAPDH as control. Data are presented as mean + SD (n = 3). (B) ABCC4 protein levels in 3T3-L1 cells at indicated phases of differentiation. Protein levels were normalized relative to GAPDH as control. Data are presented as mean + SD (n = 3). One-way ANOVA followed by the Dunnett test was performed. Asterisks represent significant p values. *p ≤ 0.05 and **p ≤ 0.01 were considered statistically significant.

FIGURE 2

ABCC4 gene silencing by siRNA. (A) Workflow for multiple siRNA treatments at 25 nM each during 3T3-L1 cell differentiation. (B) Normalized ABCC4 gene and protein expression levels post multiple siRNA treatment. Both gene and protein level expression data sets were normalized against GAPDH as control. Data are presented as mean + SD (n = 3). An unpaired t-test was performed. Asterisks represent significant p values. *p ≤ 0.05 and **p ≤ 0.01 were considered statistically significant.

FIGURE 3

Basal expression of ABCC transporters in mature 3T3-L1 cells. Basal gene expression levels of ABCC transporters in comparison with ABCC4. Gene expressions were normalized against GAPDH as control. Data are presented as mean + SD (n = 3). One-way ANOVA followed by the Dunnett test was performed. Asterisks represent significant p values. **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001 were considered statistically significant.

FIGURE 4

Silencing effect of ABCC transporters in 3T3-L1 mature adipocytes. (A) Effect of ABCC4 silencing on ABCC1 and ABCC5. (B) Effect of ABCC1 silencing on ABCC4 and ABCC5. (C) Effect of ABCC5 silencing on ABCC1 and ABCC4. Gene expression levels were normalized with GAPDH as control. Data are presented as mean + SD (n = 3). An unpaired t-test was performed. Asterisks represent significant p values. *p ≤ 0.05, ** p ≤ 0.01 and ***p ≤ 0.001 were considered statistically significant.

FIGURE 5

ABCC4 and ABCC5 knockdown promotes lipid recruitment. (A) Brightfield images of Oil red O staining of 3T3-L1 cells post silencing (Images taken at ×100 magnification). The arrow indicates the lipid droplets. The scale bar represents 50 μM. (B) Data represent lipid droplets’ optical density (OD) values following Oil red O staining. Data are presented as mean + SD (n = 3). One-way ANOVA followed by the Dunnett test was performed. Asterisks represent significant p values. ***p ≤ 0.001 was considered statistically significant.

FIGURE 6

Effect of ABCC4 and ABCC5 siRNA knockdown on genes associated with adipogenesis. PPARγ-Peroxisome proliferator-activated receptor gamma, LPL-Lipoprotein lipase FABP4-Fatty acid binding protein 4, C/EBPα-CCAAT/enhancer-binding protein-alpha, PGC-1α-Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha, ATGL- Adipose triglyceride lipase, GLUT4- Glucose transporter type 4, CD36 ⁻ Cluster of differentiation 36. Effect ABCC silencing on adipogenic genes. Gene expression levels were normalized with GAPDH as control. Data are presented as mean + SD (n = 3). One-way ANOVA followed by the Dunnett test was performed. Asterisks represent significant p values. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001 were considered as statistically significant.

FIGURE 7

ABCC4 and ABCC5 silencing promotes adipogenesis in 3T3-L1 differentiated cells. The deficiency of ABCC4 and ABCC5 function alters cAMP and PGE2 levels in intra- and extracellular compartments, respectively. (A) Intracellular cAMP levels in 3T3-L1 cells treated with negative control or forskolin or ABCC4 and ABCC5 siRNA. (B) Intracellular PGE2 levels in 3T3-L1 cells treated with negative control or ABCC4 and ABCC5 siRNA. Data are presented as mean + SD (n = 3). One-way ANOVA followed by the Bonferroni (for cAMP) and Dunnett (for PGE2) were performed. Asterisks represent significant p values. *p ≤ 0.05 and ***p ≤ 0.001 were considered as statistically significant.