Adipose tissue ATP binding cassette transporter A1 contributes to high-density lipoprotein biogenesis in vivo

Abstract

Background: Adipose tissue (AT) is the body's largest free cholesterol reservoir and abundantly expresses ATP binding cassette transporter A1 (ABCA1), a key cholesterol transporter for high-density lipoprotein (HDL) biogenesis. However, the extent to which AT ABCA1 expression contributes to HDL biogenesis in vivo is unknown.

Methods and results: Adipocyte-specific ABCA1 knockout mice (ABCA1(-A/-A)) were generated by crossing ABCA1(floxed) mice with aP2Cre transgenic mice. AT from ABCA1(-A/-A) mice had <10% of wild-type ABCA1 protein expression but normal hepatic and intestinal expression. Deletion of adipocyte ABCA1 resulted in a significant decrease in plasma HDL cholesterol (approximately 15%) and apolipoprotein A-I (approximately 13%) concentrations. AT from ABCA1(-A/-A) mice had a 2-fold increase in free cholesterol content compared with wild-type mice and failed to efflux cholesterol to apolipoprotein A-I. However, cholesterol efflux from AT to plasma HDL was similar for both genotypes of mice. Incubation of wild-type AT explants with apolipoprotein A-I resulted in the formation of multiple discrete-sized nascent HDL particles ranging in diameter from 7.1 to 12 nm; similar incubations with ABCA1(-A/-A) AT explants resulted in nascent HDL <8 nm. Plasma decay and tissue uptake of wild-type (125)I-HDL tracer were similar in both genotypes of recipient mice, suggesting that adipocyte ABCA1 deficiency reduces plasma HDL concentrations solely by reducing nascent HDL particle formation.

Conclusions: We provide in vivo evidence that AT ABCA1-dependent cholesterol efflux and nascent HDL particle formation contribute to systemic HDL biogenesis and that AT ABCA1 expression plays an important role in adipocyte cholesterol homeostasis.

Figure 1. Adipocytes abundantly express ABCA1 and efficiently produce nascent HDL particles

A. ABCA1, SR-BI, and apoE protein expression by Western blot analysis in different fat depots after 8 wks of HF diet in C57BL/6 mice (n=2 for each depot). GAPDH was used as a load control. B. Association between ABCA1 expression and FC content among six fat depots in C57BL/6 mice fed a HF diet for 8 wks (n=3 mice for each depot). Across depots, the line of best fit (r²=0.34), determined by regression analysis for all data points controlling for intra-animal correlations, is shown (p<0.01). Within depots, the overall association did not reached statistical significance due to one outlier value in retrorenal fat. C. ABCA1 expression in adipose tissue (AT), adipocytes (Adi), and stromal vascular (SV) cells. Collagenase-treated epididymal fat was fractionated into floating adipocytes (Adi) and pelleted stromal vascular cells (SV) for Western blot analysis of ABCA1 and aP2 expression. β-actin was used as a load control. D. ABCA1 expression at 0, 3, and 10 days after differentiation of SV cells into adipocytes. β-actin was used as a load control. E. Electrophoretic mobility of [¹²⁵I]-apoA-I and conditioned medium from epididymal fat tissue (Epi Fat) incubated with [¹²⁵I]-apoA-I for 24 hr on a Paragon lipoprotein agarose gel electrophoresis system. After electrophoresis, [¹²⁵I]-apoA-I mobility was visualize using a phosphorimager. α, preβ and β positions on the gel are shown for reference. F. Nascent HDL formation from epididymal tissue explants. [¹²⁵I]-apoA-I was incubated with either HEK293-ABCA1 cells (■) or 0.2 g of epididymal fat explants (○) for 24 h. Conditioned medium was then fractionated by high resolution FPLC and radiolabel in each fraction determined by gamma counting. Nascent HDL particles of increasing size are designated as preβ 1–5 based on previous studies. Elution position of [¹²⁵I]-apoA-I is shown for reference.

Figure 2. Tissue-specific expression of ABCA1 in adipocyte-specific ABCA1 knockout mice

A. ABCA1 expression in epididymal AT from WT (+/+), heterozygous (−A/+), and homozygous (−A/−A) ABCA1 knockout mice. Left panel: Western blot analysis of ABCA1, PPARγ 2 expression. ns=non-specific band. Right panel: mRNA expression of ABCA1 normalized to cyclophilin, determined by quantitative real time PCR. Mean ± SEM (n=3). B. Western blot analysis of hepatic and intestinal ABCA1 expression. ***=p<0.001. C. Western blot analysis of macrophage ABCA1 expression in resident peritoneal and bone marrow-derived macrophages (Mφ). D. Relative expression of ABCA1, aP2, and SR-BI in different tissues (i.e., liver, thioglycolate-elicited peritoneal macrophages, and 3 fat depots) of WT and ABCA1^1A/−A mice. Male mice were used in all assays and each lane represents for individual mouse. GAPDH was used as a load control in each blot.

Figure 3. Deletion of adipocyte ABCA1 reduces plasma cholesterol and HDL concentration

A. Total plasma cholesterol levels of chow-fed WT (+/+), heterozygous (−A/+) and homozygous (−A/−A) adipocyte-specific ABCA1 knockout mice. B. Plasma levels of apoA-I, apoE, and LCAT determined by Western blot analysis of 1 ul of plasma from four chow-fed WT and ABCA1^−A/−A mice. C. Total plasma cholesterol concentration of WT (+/+; ○) and ABCA1^−A/−A (A/−A; ●) mice over 16 wks of HF-diet feeding (2-way ANOVA with repeated measures; time effect, p<0.0001, genotype effect, p<0.001; n=4 of each genotype). D. FPLC separation of plasma lipoproteins from WT (+/+; ○) and ABCA1^−A/−A (−A/−A; ●) mice before (chow) and after 2 and 16 wks of HF diet feeding. Each profile represents a separation of a plasma pool from four mice of each genotype. Lipoprotein elution was monitored by enzymatic assay of cholesterol in individual fractions (Fr); elution position of VLDL, LDL, and HDL are indicated in the left panel.

Figure 4. Deletion of adipocyte ABCA1 does not affect HDL catabolism

A. HDL isolated from WT mice was radiolabeled with ¹²⁵I-tyramine cellobiose (TC) and injected into the jugular vein of +/+ (WT; n=4) and −A/−A (ABCA1^−A/−A; n=4) recipient mice. Periodic blood samples over a 24 h time period after injection were withdrawn to quantify the radioactivity remaining in plasma, normalized to percentage of injected tracer. B. Twenty-four hr after ¹²⁵I-TC-HDL tracer injection, mice were sacrificed and liver, kidney, and epididymal fat (adipose) were removed to quantify tracer uptake by each organ. Data were normalized to liver uptake in wild type mice.

Figure 5. Adipocyte ABCA1 generates heterogeneous-sized nascent HDL particles

A. Free cholesterol accumulation in epididymal AT with progressive inactivation of adipocyte ABCA1 alleles. WT (+/+), heterozygote (−A/+), and homozygote (−A/−A) of adipocyte-specific ABCA1 knockout mice. B. Cholesterol efflux to apoA-I (ABCA1 dependent) and HDL (ABCA1 independent). Ex vivo cultures of epididymal fat from +/+ and −A/−A mice were radiolabeled with ³H-cholesterol for 24 h, washed, and then incubated with no addition (no Trt), apoA-I (hAI; 20 ug/ml), or mouse HDL (mHDL; 50 ug/ml) for 4 hr to measure ³H-FC efflux into the medium. For panels A and B, mean ± SEM, n=4. * p<0.05, ** p<0.01, ***p<0.001, ns= not significant at p=0.05. C. Nascent HDL formation in the presence of ¹²⁵I-apoA-I in ex vivo cultures of epididymal AT from +/+ and −A/−A mice. After a 24 h incubation, conditioned medium was fractionated by high resolution FPLC and radiolabeled apoA-I in each fraction was quantified using a gamma counter. Elution regions for different-sized nascent pre-β HDL particles are denoted by the vertical dashed lines. Inset shows the size of AT derived-nascent HDLs separated by non-denaturating gradient gel electrophoresis (4–30%). Migration positions of the radiolabeled nascent HDL particles were visualized using a phosphorimager.