ABCD2 is abundant in adipose tissue and opposes the accumulation of dietary erucic acid (C22:1) in fat

Abstract

The ATP binding cassette transporter, ABCD2 (D2), is a peroxisomal protein whose mRNA has been detected in the adrenal, brain, liver, and fat. Although the role of this transporter in neural tissues has been studied, its function in adipose tissue remains unexplored. The level of immunoreactive D2 in epididymal fat is >50-fold of that found in brain or adrenal. D2 is highly enriched in adipocytes and is upregulated during adipogenesis but is not essential for adipocyte differentiation or lipid accumulation in day 13.5 mouse embryonic fibroblasts isolated from D2-deficient (D2(-/-)) mice. Although no differences were appreciated in differentiation percentage, total lipid accumulation was greater in D2(-/-) adipocytes compared with the wild type. These results were consistent with in vivo observations in which no significant differences in adiposity or adipocyte diameter between wild-type and D2(-/-) mice were observed. D2(-/-) adipose tissue showed an increase in the abundance of 20:1 and 22:1 fatty acids. When mice were challenged with a diet enriched in erucic acid (22:1), this lipid accumulated in the adipose tissue in a gene-dosage-dependent manner. In conclusion, D2 is a sterol regulatory element binding protein target gene that is highly abundant in fat and opposes the accumulation of dietary lipids generally absent from the triglyceride storage pool within adipose tissue.

Fig. 1.

Relative expression of immunoreactive D2 in selected mouse tissues. Total membrane proteins were prepared from tissues of male C57Bl6/J mice. Equal amounts of proteins were pooled from each mouse and analyzed by immunoblotting using the antibody directed against D2. Specificity of the immunoreactive D2 bands was confirmed using tissues from D2-deficient mice (D2^−/−) mice. Epi., epididymal; NSB, nonspecific band.

Fig. 2.

Relative expression of D2 mRNA and protein among selected mouse fat depots. A: The relative levels of D2 and D1 mRNA among fat pads were measured by qRT-PCR and normalized to cyclophilin and epididymal fat using the ΔΔCT method. B: Total membrane proteins were prepared from epididymal (Epi.), inguinal, retroperitoneal, mesenteric, and brown fat pads and analyzed by immunoblotting. Epididymal and brown fat pads from D2^−/− mice were also analyzed to ensure specificity of immunoreactive proteins. The blot was reprobed for calnexin to verify equal loading of membrane proteins. Asterisk denotes residual signal from D2 immunoblot upon reblotting with calnexin.

Fig. 3.

Relative expression of D2 mRNA and protein among adipose tissue fractions. A: Adipocytes were separated from stromal vascular (SV) cells by collagenase digestion and low-speed centrifugation. The mRNA levels of D2 were measured by qRT-PCR as described in Fig. 2. FAS and ACC were used as positive controls for adipocytes. Macrophage (CD68) and endothelial cells (CD31) were used as controls for separation of stromal vascular cells from adipocytes. B: Total membranes were prepared and analyzed by immunoblotting. CD36 was used as a control, although it is somewhat enriched in adipocytes.

Fig. 4.

Expression of D2 mRNA and protein during adipogenesis in 3T3-L1 cells. A: 3T3-L1 cells were differentiated to adipocytes and stained with Oil-Red O. B: D2 mRNA levels were determined on even days of differentiation by qRT-PCR. Fatty acid binding protein 4 (aP2) was examined as positive control for differentiation. D1 was examined as negative control. C: D2 protein expression during differentiation was analyzed by immunoblotting. β-Actin was blotted as loading control. Asterisk denotes the nonspecific band.

Fig. 5.

Fatty acid profile in adipose tissue from wide-type, heterozygous (D2^+/−), and D2^−/− mice (n = 4) fed with an erucic acid enriched diet. A: Total lipids were extracted from adipose and analyzed by GC-MS-FID. Fatty acid abundance was normalized to C17 internal standard and expressed milligram/milligram of white adipose tissue mass (WAT). B: Fatty acid profile of serum from mice maintained on an erucic acid diet and fasted for 24 h. Total serum lipids were extracted with Folch reagent and prepared for GC-MS-FID as described for adipose tissue. Inset: total free fatty acid in serum following a 24 h fast. Asterisks denote significant differences from the wild type (P < 0.05).