Background Cholesterol efflux capacity is a tissue culture assay for HDL function that is not amenable for high-throughput monitoring of risk assessment. Methods We devised a cell-free HDL function assay to measure the exchange rate of exogenous apoA1 into serum HDL using NBD/Alexa647 double-labeled apoA1, whose NBD/Alexa647 emission ratio increased upon exchange into HDL. ApoA1 exchange rate (AER) was assayed by incubating labeled apoA1 with human serum, and the rate of the increase of the NBD/Alexa647 ratio over time was calculated as AER. Results Fast protein liquid chromatography analysis of serum confirmed that the labeled apoA1 selectively exchanged into the HDL lipoprotein fraction. Characterization studies demonstrated that the AER assay had excellent intra- and inter-day reproducibility, was stable over 3 freeze-thaw cycles, and yielded similar results with serum or plasma. We quantified AER in serum from randomly selected stable subjects undergoing elective diagnostic coronary angiography (n = 997). AER was correlated with HDL-cholesterol (r = 0.58, P < 0.0001) and apoA1 levels (r = 0.56, P < 0.0001). Kaplan-Meier survival plot showed subjects in the lowest quartile of AER experienced a significantly higher rate of incident major adverse cardiovascular events (MACE = myocardial infarction, stroke, or death) (P < 0.0069 log rank). Moreover, compared to subjects in the lowest AER quartile, the remaining subjects showed significantly lower incident (3 year) risk for MACE, even after adjustment for traditional risk factors and apoA1 (HR 0.58; 95% CI 0.40–0.85; P = 0.005). Conclusions In a prospective cohort of stable subjects undergoing elective diagnostic cardiac evaluations, low AER was associated with increased incident risk of MACE.

Altered lipid metabolism and inflammation are involved in the pathogenesis of both nonalcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD). Even though high-density lipoprotein (HDL), a CVD protective marker, is decreased, whether HDL metabolism and function are perturbed in NAFLD are currently unknown. We examined the effect of NAFLD and disease severity on HDL metabolism and function in patients with biopsy-proven simple steatosis (SS), nonalcoholic steatohepatitis (NASH), and healthy controls. HDL turnover and HDL protein dynamics in SS ( n = 7), NASH ( n = 8), and healthy controls ( n = 9) were studied in vivo. HDL maturation and remodeling, antioxidant, cholesterol efflux properties, and activities of lecithin-cholesterol ester acyltransferase and cholesterol ester transfer protein (CETP) were quantified using in vitro assays. All patients with NAFLD had increased turnover of both HDL cholesterol (HDLc; 0.16 ± 0.09 vs. 0.34 ± 0.18 days, P < 0.05) and apolipoprotein A1 (ApoAI) (0.26 ± 0.04 vs. 0.34 ± 0.06 days, P < 0.005) compared with healthy controls. The fractional catabolic rates of other HDL proteins, including ApoAII (and ApoAIV) were higher ( P < 0.05) in patients with NAFLD who also had higher CETP activity, ApoAI/HDLc ratio ( P < 0.05). NAFLD-induced alterations were associated with lower antioxidant (114.2 ± 46.6 vs. 220.5 ± 48.2 nmol·mL−1·min−1) but higher total efflux properties of HDL (23.4 ± 1.3% vs. 25.5 ± 2.3%) (both P < 0.05), which was more pronounced in individuals with NASH. However, no differences were observed in either HDL turnover, antioxidant, and cholesterol efflux functions of HDL or HDL proteins’ turnover between subjects with SS and subjects with NASH. Thus, HDL metabolism and function are altered in NAFLD without any significant differences between SS and NASH.

Bariatric surgery improves glycemic control better than medical therapy; however, the effect of bariatric surgery on HDL function is not well characterized. Serum samples were available at baseline, 1-, and 5-years post procedures, for 90 patients with obesity and type 2 diabetes who were randomized to intensive medical therapy (n = 20), Roux-en-Y gastric bypass (RYGB, n = 37), or sleeve gastrectomy (SG, n = 33) as part of the STAMPEDE clinical trial. We examined serum HDL function by two independent assays, apolipoprotein A-1 exchange rate (AER) and cholesterol efflux capacity (CEC). Compared with baseline, AER was significantly higher at 5 years for participants in all treatment groups, but only increased significantly at 1 year in the RYGB and SG groups. CEC was divided into ABCA1-dependent and independent fractions, and the later was correlated with AER. ABCA1-independent CEC increased significantly only at 5 years in both surgical groups, but did not significantly change in the medical therapy group. There was no significant change in ABCA1-dependent CEC in any group. The increase in AER, but not ABCA1-independent CEC, was correlated with the reduction in body mass index and glycated hemoglobin levels among all subjects at 5 years, indicating that AER as a measure of HDL function would be a better reflection of therapy versus CEC.

High-density lipoprotein (HDL) metabolism is facilitated in part by scavenger receptor class B, type 1 (SR-B1) that mediates HDL uptake into cells. Higher levels of HDL have been associated with protection in other diseases, however, its role in prostate cancer is not definitive. SR-B1 is up-regulated in prostate cancer tissue, suggesting a possible role of this receptor in tumor progression. Here, we report that knockout (KO) of SR-B1 in both human and mouse prostate cancer cell lines through CRISPR/Cas9-mediated genome editing reduces HDL uptake into the prostate cancer cells and reduces their proliferation in response to HDL. In vivo studies using syngeneic SR-B1 WT (SR-B1+/+) and SR-B1 KO (SR-B1−/−) prostate cancer cells in WT and apolipoprotein-AI KO (apoA1-KO) C57BL/6J mice revealed that WT hosts, containing higher levels of total and HDL-cholesterol, grew larger tumors than apoA1-KO hosts with lower levels of total and HDL-cholesterol. Furthermore, SR-B1−/− prostate cancer cells formed smaller tumors in WT hosts than SR-B1+/+ cells in the same host model. Increased tumor volume was overall associated with reduced survival. We conclude that knocking out SR-B1 in prostate cancer tumors reduces HDL-associated increases in prostate cancer cell proliferation and disease progression.

We have previously shown that the DBA/2J versus AKR/J mouse strain is associated with decreased autophagy-mediated lysosomal hydrolysis of cholesterol esters. Our objective was to determine differences in lysosome function in AKR/J and DBA/2J macrophages, and identify the responsible genes. Using a novel dual-labeled indicator of lysosome function, DBA/2J versus AKR/J bone marrow derived macrophages had significantly decreased lysosome function. We performed quantitative trait loci mapping of lysosome function in bone marrow macrophages from an AKR/J × DBA/2J strain intercross. Four distinct lysosome function loci were identified, which we named macrophage lysosome function modifier (Mlfm) Mlfm1 through Mlfm4. The strongest locus Mlfm1 harbors the Gpnmb gene, which has been shown to recruit autophagy protein light chain 3 to autophagosomes for lysosome fusion. The parental DBA/2J strain has a nonsense variant in Gpnmb. siRNA knockdown of Gpnmb in AKR/J macrophages decreased lysosome function, and Gpnmb deletion through CRISP/Cas9 editing in RAW 264.7 mouse macrophages also demonstrated a similar result. Furthermore, a DBA/2 substrain, called DBA/2J-Gpnmb+/SjJ, contains the wildtype Gpnmb gene, and macrophages from this Gpnmb-preserved DBA/2 substrain exhibited recovered lysosome function. In conclusion, we identified Gpnmb as a causal modifier gene of lysosome function in this strain pair.