Adipocyte modulation of high-density lipoprotein cholesterol

Abstract

Background: Adipose harbors a large depot of free cholesterol. However, a role for adipose in cholesterol lipidation of high-density lipoprotein (HDL) in vivo is not established. We present the first evidence that adipocytes support transfer of cholesterol to HDL in vivo as well as in vitro and implicate ATP-binding cassette subfamily A member 1 (ABCA1) and scavenger receptor class B type I (SR-BI), but not ATP-binding cassette subfamily G member 1 (ABCG1), cholesterol transporters in this process.

Methods and results: Cholesterol efflux from wild-type, ABCA1(-/-), SR-BI(-/-), and ABCG1(-/-) adipocytes to apolipoprotein A-I (apoA-I) and HDL3 were measured in vitro. 3T3L1 adipocytes, labeled with (3)H-cholesterol, were injected intraperitoneally into wild-type, apoA-I transgenic, and apoA-I(-/-) mice, and tracer movement onto plasma HDL was monitored. Identical studies were performed with labeled wild-type, ABCA1(-/-), or SR-BI(-/-) mouse embryonic fibroblast adipocytes. The effect of tumor necrosis factor-alpha on transporter expression and cholesterol efflux was monitored during adipocyte differentiation. Cholesterol efflux to apoA-I and HDL3 was impaired in ABCA1(-/-) and SR-BI(-/-) adipocytes, respectively, with no effect observed in ABCG1(-/-) adipocytes. Intraperitoneal injection of labeled 3T3L1 adipocytes resulted in increased HDL-associated (3)H-cholesterol in apoA-I transgenic mice but reduced levels in apoA-I(-/-) animals. Intraperitoneal injection of labeled ABCA1(-/-) or SR-BI(-/-) adipocytes reduced plasma counts relative to their respective controls. Tumor necrosis factor-alpha reduced both ABCA1 and SR-BI expression and impaired cholesterol efflux from partially differentiated adipocytes.

Conclusions: These data suggest a novel metabolic function of adipocytes in promoting cholesterol transfer to HDL in vivo and implicate adipocyte SR-BI and ABCA1, but not ABCG1, in this process. Furthermore, adipocyte modulation of HDL may be impaired in adipose inflammatory disease states such as type 2 diabetes mellitus.

Figure 1

Mouse embryonic fibroblasts (MEF), derived from wild-type (WT), ABCA1−/− or SRB1−/− mice were differentiated and labeled overnight with ³H-cholesterol (5µCi/mL). Cells were equilibrated and treated overnight±LXR agonist (10µM) and co-treated±LXR agonist, ±BLT (10µM) or ±Probucol (20µM) for 2h. Cholesterol efflux from WT (A and B), WT and ABCA1−/− (C and D), and WT and SR-BI−/− (E and F) MEF adipocytes to apoA-I (20µg/ml) and HDL3 (50µg/ml) over 4h is presented; background efflux to MEM was subtracted. Efflux is presented as % total ³H-cholesterol loaded into cells (n=3–4, *p<0.05, **p<0.01, ***p<0.001 vs. control).

Figure 2

MEF cells derived from ABCG1 or WT mice were differentiated and labeled overnight with ³H-cholesterol (5µCi/mL), equilibrated and then washed with PBS and efflux to apoA-I (20µg/ml), HDL3 (50µg/ml) or 5% serum was monitored over 4h. RNA and protein was extracted. (A) Efflux from WT and ABCG1−/− to ApoA-I, HDL3 and serum. (B) ABCG1 mRNA levels were markedly reduced in ABCG1−/− cells compared with WT control (n=3, ***p<0.001). (C) ABCG1 protein levels were barely detectable in WT and ABCG1−/− MEF adipocytes compared with positive control mouse liver lysate, positive control.

Figure 3

3T3L1 adipocytes were labeled with ³H-cholesterol (5µCi/mL) overnight and equilibrated for 24h. Equal numbers of adipocytes (in 0.5mL) were intraperitoneally (IP)-injected into apoA-I transgenic (solid circle), apoA-I−/− (solid square) or wild-type (open circle) mice. Movement of ³H-cholesterol from IP-injected 3T3L1 adipocytes into (A) plasma and (B) feces was monitored over 48h (n=6, *p<0.05, **p<0.01, ***p<0.001 vs. wild-type animals). Pooled plasma was separated by FPLC and levels of (C) cholesterol mass and (D) ³H-cholesterol tracer was measured.

Figure 4

ABCA1−/− (A&B) or SR-BI−/− (C&D) MEF adipocytes and litter-mate wild-Type (WT) control MEF adipocytes were labeled with ³H-cholesterol (5µCi/mL) overnight and equilibrated. C57BL/6 WT mice were injected IP with WT (solid circle) or ABCA1−/− (open circle) MEF-adipocytes and levels of ³H-cholesterol tracer in (A) plasma and (B) feces was measured over 48h. C57BL/6 WT mice were injected with WT (solid circle) or SR-BI−/− (open circle) MEF-adipocytes and levels of ³H-cholesterol tracer in (C) plasma and (D) feces was measured. Levels of ³H-cholesterol are presented as % total cpm injected (n=12, *p<0.05, **p<0.01 and ***p<0.001 vs. animals injected with WT adipocytes).

Figure 5

The effect of TNFα (10ng/ml) on cholesterol efflux from 3T3L1 cells during differentiation was assessed at day 0 (pre-adipocyte), day 5 (partially-differentiated) and day 10 (mature). Cells were labeled with ³H-cholesterol overnight prior to equilibration ±TNFα (10ng/ml). Efflux to (A) apoA-I (20µg/ml) and (B) HDL3 (50µg/ml) at day 0, 5 and 10 ±TNFα are presented. (C) Effects of TNFα vs. saline on ABCA1, ABCG1 and SR-BI protein levels at day 0, 5 and 10.

Figure 6

As adipose inflammation is a hallmark of central obesity and type-2 diabetes, loss of adipocyte lipidation of HDL may directly contribute to lower HDL-C levels in these inflammatory, insulin resistant states. Despite greater adipose mass and cholesterol content in adiposity, adipocyte inflammation is associated with reduced expression of the cholesterol efflux transporters, ABCA1 and SR-BI, and impaired cholesterol efflux to apoA-I and HDL particles.