Table 1.
Plasma and liver lipids.
Fig 1.
Radiolabelled cholesterol accumulation in the plasma, liver and adipose tissue.
The concentration of radiolabelled cholesterol as percent of injected dose in (A) plasma over 48 h, (B) per gram of liver wet weight, (C) total liver protein, (D) per gram of adipose wet weight and (E) total adipose mass. A known volume of plasma (30 mL) was measured directly using liquid scintillation counting while lipids were extracted from tissue by Bligh-Dyer before quantifying. Values were then expressed as a % of the total amount of radiolabelled cholesterol injected. *p<0.0005, **p = 0.03.
Fig 2.
Radiolabelled cholesterol accumulation in the bile and faeces.
The amount of cholesterol accumulation in the bile (A) was determined by measuring a known volume by liquid scintillation counting. Cholesterol accumulation in the faeces (B) as was determined by lipid extraction using the Bligh-Dyer method and then quantified by liquid scintillation counting. Both measures were then expressed as a % of the total amount of radiolabelled cholesterol injected. *p = 0.002; **p<0.0001.
Fig 3.
Western blot for SR-BI, ABCA1, ABCG1 and ABCG5 protein expression.
Liver (A) and adipose (B) tissue were homogenised into lysates in RIPA buffer with protease inhibitors and PMSF. The lysates (10–20 μg protein for liver and 40 μg protein for adipose) were loaded onto a 3–12% SDS gel and transferred onto nitrocellulose membrane. The membranes were probed for SR-BI, ABCA1, ABCG1 and ABCG5 (liver only) and visualised with ECL and densitometry. Results are normalised to the α-tubulin loading control. n = 3–4 mice of each genotype; *p<0.01.
Fig 4.
Cholesterol efflux potential of the serum and adipose and liver ex vivo cholesterol efflux capacity and cholesterol uptake of ob/ob and wt mice.
Serum (A) was obtained from the controls (white bars) and ob/ob (black bars) mice, prior to the RCT procedure and used at 2.0% concentration on a set of cells expressed ABCA1, ABCG1 and SR-BI. The cells were prelabelled with [3H]-cholesterol and incubated with the serum for 4 h before the amount of radiolabel was determined in the media by liquid scintillation counting. Percent cholesterol efflux was determined as concentration of label in the media as a function of total label in the cells. Cellular cholesterol efflux was also quantified using the apoB-depleted fraction of the serum (B) where serum was incubated with PEG (2:5), pelleted and then the supernatant used at 2.4% v/v. Freshly isolated adipose (C) and liver tissue (D) were cut into small pieces, cholesterol-loaded with [3H]-cholesterol and incubated for 4 h with cholesterol acceptors apoA-I and HDL or media (passive) to measure adipose and liver ex vivo efflux capacity. Freshly isolated adipose (E) and liver tissue (F) were also used to measure cholesterol uptake by measuring the amount of [3H]-cholesterol taken up over a 24 h period. Lipids were extracted from the tissue with isopropanol and [3H]-cholesterol quantified by liquid scintillation counting, which was then normalised to protein concentration in each well. RCT, reverse cholesterol transport; SR-BI, scavenger receptor class B type 1; ABCG1, ATP-binding cassette sub-family G member 1; PEG, polyethylene glycol. *p<0.05; **p<0.002; ***p<0.00005;****p<0.003.
Fig 5.
Cellular cholesterol efflux from bone marrow-derived macrophages.
The bone marrow of the littermate (white bars) and ob/ob (black bars) animals was harvested from the femur of 6 week old male mice and differentiated into macrophages using L-cell media. The cholesterol effluxing ability of the BMM were quantified using 2.0% whole serum, HDL (50 mg/ml) and apoA-I (10 mg/ml). The BMDM were tested when unloaded (A) and also after loaded (B) with addition of 100 mg/ml of acLDL during the radiolabelling period of the cells. BMDM, bone marrow-derived macrophages; acLDL, acetylated LDL.
Fig 6.
Schematic of RCT in the ob/ob mouse.
The movement of cholesterol from the macrophage into blood is via ABCA1-, ABCG1- and SR-BI-mediated cholesterol efflux and this is unaffected in the ob/ob mice. Transport of the cholesterol throughout the body is mainly done by lipoproteins and in particular, HDL. However, uptake of cholesterol by the liver and adipocytes is compromised resulting in longer retention time in the blood and slower clearance of the cholesterol from the body via the bile and faeces. The HDL and serum of ob/ob mice have enhanced cholesterol efflux capacity which may have contribute further to the increase in plasm cholesterol.