10,12-Conjugated linoleic acid supplementation improves HDL composition and function in mice

Abstract

Obesity is associated with inflammation, insulin resistance, and type 2 diabetes, which are major risk factors for CVD. One dietary component of ruminant animal foods, 10,12-conjugated linoleic acid (10,12 CLA), has been shown to promote weight loss in humans. Previous work has shown that 10,12 CLA is atheroprotective in mice by a mechanism that may be distinct from its weight loss effects, but this exact mechanism is unclear. To investigate this, we evaluated HDL composition and function in obese LDL receptor (Ldlr^-/-) mice that were losing weight because of 10,12 CLA supplementation or caloric restriction (CR; weight-matched control group) and in an obese control group consuming a high-fat high-sucrose diet. We show that 10,12 CLA-HDL exerted a stronger anti-inflammatory effect than CR- or high-fat high-sucrose-HDL in cultured adipocytes. Furthermore, the 10,12 CLA-HDL particle (HDL-P) concentration was higher, attributed to more medium- and large-sized HDL-Ps. Passive cholesterol efflux capacity of 10,12 CLA-HDL was elevated, as was expression of HDL receptor scavenger receptor class B type 1 in the aortic arch. Murine macrophages treated with 10,12 CLA in vitro exhibited increased expression of cholesterol transporters Abca1 and Abcg1, suggesting increased cholesterol efflux potential of these cells. Finally, proteomics analysis revealed elevated Apoa1 content in 10,12 CLA-HDL-Ps, consistent with a higher particle concentration, and particles were also enriched with alpha-1-antitrypsin, an emerging anti-inflammatory and antiatherosclerotic HDL-associated protein. We conclude that 10,12 CLA may therefore exert its atheroprotective effects by increasing HDL-P concentration, HDL anti-inflammatory potential, and promoting beneficial effects on cholesterol efflux.

Keywords: Abca1; HDL particle concentration; HDL particle size; HDL proteomics; alpha-1-antitrypsin; cholesterol transporters; fast-phase liquid chromatography; scavenger receptor class B member 1; serum amyloid A; weight loss.

Fig. 1

Mouse experimental design. Male Ldlr^−/− mice (10 weeks of age) were fed an HFHS diet for 12 weeks, then continued on the HFHS diet for an additional 8 weeks with the following: 1) no variations (mice continued on the HFHS diet); 2) HFHS diet with 1% added 10,12 CLA; and 3) HFHS diet plus CR to match the level of weight loss achieved by mice supplemented with 10,12 CLA. Blood was collected for HDL isolation to determine HDL-P number and size distribution, proteomics, cholesterol efflux capacity, and anti-inflammatory capacity. n = 8 mice/group.

Fig. 2

HDL anti-inflammatory assay using cultured adipocytes. Fully differentiated 3T3-L1 adipocytes were pretreated with the indicated mouse and hHDL preparations (50 μg/ml) for 6 h, cells were thoroughly washed three times, then treated with PA (250 μM) for 24 h. Expression levels of genes indicative of inflammation including Saa3, Ccl2, and Il1b were measured and normalized to B2m, presented relative to untreated cells (media). Different letters indicate significant differences, assessed using one-way ANOVA with multiple comparisons (Tukey) (P < 0.05). cHDL, control HDL from lean chow-fed mice; HFHS + CR-HDL, HDL from mice fed a HFHS diet that were calorically restricted; HFHS-HDL, HDL from mice fed an HFHS diet; HFHS + 10,12-HDL, HDL from mice fed an HFHS diet containing 10,12 CLA; hHDL, human HDL from healthy lean subjects; snHDL, HDL from mice injected with silver nitrate.

Fig. 3

HDL-P size analysis and FPLC. A: Plasma cholesterol and triglycerides. B: Pooled plasma samples from each treatment group were separated by FPLC. Areas under the curve for fractions containing HDL (30, 31, 32, 33, 34, 35) are presented. C: Total, medium (M), large (L), and extralarge (XL) HDL-P concentrations were determined using calibrated-ion mobility analysis. D: Liver Lcat mRNA expression, presented normalized to Gapdh. n = 8 mice/group; ∗P < 0.05. FPLC, fast-phase liquid chromatography.

Fig. 4

Cholesterol efflux capacity and cholesterol transporter expression. A: Cholesterol efflux capacity (CEC) of the indicated HDL preparations was quantified from radiolabeled cholesterol-loaded J774 macrophages. Total CEC is the sum of basal (Abca1-independent) and Abca1-depenent efflux (determined by pretreatment with a cAMP agonist). B: Cholesterol transporter gene expression from aortic arch and the immediately adjacent PVAT. C: Cholesterol transporter gene (left panel) and protein expression (middle and right panels) from BMDM cultured in the presence or the absence of 100 μM 10,12 CLA or 9,11 CLA (inert control), with or without an LXR agonist (T0901317, 5 μg/ml) for 24 h; n = 8 mice/group; ∗P < 0.05.

Fig. 5

Proteomics confirmation and hepatic gene expression. A: HDL preparations from the indicated treatment groups were subjected to immunoblot and probed for Saa (marker = 15 kD) and Apoa1 (marker = 35 kD). Densitometry was performed using ImageJ software. Control HDL samples were pooled; n = 3 for treatment groups. Different letters indicate significant differences, assessed using one-way ANOVA with multiple comparisons (Tukey) (P < 0.05). cH, control HDL from mice injected with saline; CR-HDL, HDL from mice fed an HFHS diet that were calorically restricted; 10,12-HDL, HDL from mice fed an HFHS diet containing 10,12 CLA; HFHS-HDL, HDL from mice fed an HFHS diet; sH, HDL from mice injected with silver nitrate. B: Liver gene expression was quantified from the indicated treatment groups. n = 8 mice/group, ∗P < 0.05 from HFHS.