Background and Aims

Increased plasma triglyceride levels are commonly observed in patients with obesity, type 2 diabetes and metabolic syndrome. The efficiency of current available treatments is however limited, and a more comprehensive understanding of the underlying pathological processes is thus demanded. Lipases and lipoproteins play a central role in maintaining a balanced triglyceride turnover. A heterogenous lipoprotein fraction carries apolipoprotein M (apoM) which in return binds Sphingosine-1-Phosphate (S1P). Both mediators play a role in energy metabolism. Their exact contribution is however elusive, and thus addressed by the present study.

Methods

We investigated by 10-fold increased plasma apoM and 1-fold increased S1P levels in a transgenic mouse model. Molecular processes related to glucose and fat metabolism were thereby emphasized.

Results

Most striking, elevated apoM/S1P levels delayed plasma triglyceride turnover by 80%. Analyses of molecular processes, centrally involved in triglyceride clearance, revealed reduced lipase activities as the primary underlying cause. Administration of FTY720, a S1P analogue, reduced the triglyceride clearance rate substantially, thus suggesting a S1P mediated effect. Changes in tissue-specific lipid uptake were additionally evident.

Conclusions

We conclude that S1P is a potent regulator of plasma triglyceride clearance, whereas apoM plays a vital role in S1P transport. In line with previous studies, the apoM/S1P system is thus a considerable target to reduce plasma triglycerides in human subjects.

Background and Aims

Triglyceride-rich lipoproteins (TRLs) have gained renewed attention as Mendelian randomization studies have verified their causal role in human atherosclerotic disease. Lipoprotein lipase (LPL) is a key enzyme in TRL metabolism and facilitates hydrolysis of circulating TRLs. The LPL regulatory system consists of several complex and dynamic mechanisms. Different proteins may inhibit or promote LPL activity at different stages: from protein synthesis and folding to transport and final hydrolytic action at the capillary lumen.

Previous studies of LPL regulation have mainly been performed in non-physiological settings, using knockout/overexpression in animal models, or by in vitro experiments adding supraphysiological concentrations. In this study, we wanted to examine the association between physiological LPL regulator levels and activity in the plasma compartment.

Methods

Using isothermal titration calorimetry, we performed an LPL activity assay using plasma samples from 117 fasted individuals. The assay allows direct measurement of lipoprotein hydrolysis using a standardised amount of LPL. Additionally, we measured plasma concentrations of seven known LPL regulators with ELISA. The plasma lipidome was profiled with nuclear magnetic resonance spectroscopy.

Results

Physiological variation in the levels of apolipoprotein C1, C2 or C3, and angiopoietin-like protein 3, 4 or 8 showed no significant association with LPL activity (Figure 2C). Instead, LPL activity was strongly associated with characteristics of the plasma lipidome, such as the average very low-density lipoprotein particle size and the lipid content in different lipoprotein subclasses (Figure 3A-B).

Conclusions

LPL activity in human plasma is affected by the physiological properties of the plasma rather than the levels of LPL regulators.

Background and Aims

While hypertriglyceridemic pancreatitis (HTGP) is less likely to occur with serum triglycerides (TG) < 1000 mg/dL, epidemiological studies demonstrate elevated risk even with moderate HTG. Identification of patients with less severe HTG at risk for HTGP may aide in preventing this complication. Using advanced lipoprotein testing, we examined lipid and lipoprotein profiles of HTG patients with and without HTGP to identify differences and diagnostic markers.

Methods

We retrospectively reviewed charts of adults with serum TG ≥ 500 mg/dL who had undergone advanced lipoprotein testing between 2005–2018. Chi-square or rank-sum tests were used to compare patients with and without HTGP within 1 year of testing. Receiver operating characteristic (ROC) curves were used to examine the value of lipid and lipoprotein parameters in discriminating between the two groups.

Results

Of 58 patients analyzed, 20 had at least one instance of HTGP. Median serum and chylomicron TG values were higher in HTGP patients vs. controls (2832 mg/dL vs. 978 mg/dL; 1255 mg/dL vs. 266 mg/dL respectively, p < 0.001). Significant between-group differences were noted in Chylomicron TG:Total TG, Chylomicron TG:VLDL TG and Chylomicron TG:Apo B ratios. Chylomicron TG:Apo B had the best discriminant value with an AUC of 0.83 (p<0.0001). A ratio > 2 was associated with a sensitivity of 90%, and ratio > 11.5 was associated with a specificity of 90% for predicting HTGP.

Conclusions

Marked differences in lipid and lipoprotein levels were noted in HTG patients with and without pancreatitis. Chylomicron TG:Apo B ratio is most useful in identifying those at risk for HTGP.

Background and Aims

HDL-mediated stimulation of cholesterol efflux from macrophage foam cells initiates the reverse cholesterol pathway (m-RCT) which ends in the fecal excretion of macrophage-derived unesterified cholesterol (UC). We investigated the role of LDL as an intermediate UC carrier in m-RCT.

Methods

Macrophage cholesterol efflux induced in vitro by LDL added to the culture media either alone or together with HDL, or ex vivo by plasma derived from subjects with familial hypercholesterolemia (FH) was assessed. In vivo m-RCT was evaluated in CETP lacking mouse models of hypercholesterolemia fed a Western-type diet.

Results

LDL facilitated the removal of radiolabeled UC from cultured macrophages, and, in the simultaneous presence of HDL, a rapid transfer of the radiolabeled UC from HDL to LDL occurred. However, LDL did not exert a synergistic effect on the CEC of HDL or of FH plasma. The m-RCT rates of radiolabeled UC for LDL receptor (LDLr)-KO, LDLr-KO/apoB-100, and PCSK9-overexpressing mice were all significantly attenuated compared with those of wild-type mice. In contrast, the m-RCT rate remained unchanged in apoB-100 overexpressing transgenic mice with fully functional LDLr, despite increased levels of plasma apoB-containing lipoproteins.

Conclusions

We conclude that hepatic LDLr contributes to the flow of macrophage-derived UC to feces in mice, while hyper-apoB lipoproteinemia per se is unable to further stimulate the m-RCT. The results suggest that, besides the major HDL-dependent m-RCT pathway via SR-BI to the liver, a CETP-independent m-RCT path exists in mice in which LDL mediates the transfer of UC cholesterol from macrophages to feces.

Background and Aims

Plasma high-density lipoprotein cholesterol (HDL-C) levels are inversely associated with cardiovascular disease risk. Whereas the causal nature of this relationship was challenged by genetic studies and several outcome trials, an increasing number of animal and in vitro studies emphasized the effects of HDL on the immune system. In vivo data supporting these effects on monocytes in patients are lacking. Here, we evaluated whether low levels of HDL-C determined by genetic mutations (e.g. ABCA1, APOA1 and LCAT) are associated with monocyte activation in humans.

Methods

We compared the monocyte phenotype from subjects with low-HDL (either ABCA1, apoA-I or LCAT deficient) to normolipidemic healthy controls. We used flow cytometry, measured intracellular lipid content as well as used functional read-outs such as trans-endothelial migration capacity and cytokine production. Finally, we studied RNA expression of pivotal inflammatory as well as cholesterol homeostasis genes.

Results

We found that apoA-I and ABCA1 deficiency associate with a pro-inflammatory monocyte phenotype with increased levels of activation markers, increased cytokine production and transendothelial migration capacity compared to controls. Baseline expression of IL-1b was increased. In contrast, LCAT deficiency shows an opposite phenotype with reduced transendothelial migration capacity and increased expression of efflux receptors. This is accompanied by a decreased cytokine production capacity and lower activation markers.

Conclusions

Different HDL-genotypes are associated with lead opposite monocyte immunophenotypes, resulting in either pro- or anti-inflammatory monocytes. Monocytes from HDL-deficient patients have a genotype specific pro- or anti-inflammatory phenotype and HDL-deficient patients may therefore benefit from optimization of a more personalized treatment strategy.

Background and Aims

A diet rich in polyunsaturated fatty acids (PUFAs), especially in eicosapentaenoic acid (EPA, C20:5 n-3) is cardioprotective. We investigated the impact of several PUFAs: EPA, arachidonic acid (AA, C20:4 n-6) and docosahexaenoic acid (DHA, C22:6 n-6) on ABCA1-mediated cholesterol efflux, an antiatherogenic pathway.

Methods

Human monocytic leukemia THP-1 cells differentiated into macrophages with PMA were incubated for 34 h without (control cells) or with 70 μM EPA, 50 μM AA or 15 μM DHA. After an overnight treatment with LXR/RXR agonists, the isotopic cholesterol efflux promoted by lipid-free apolipoprotein AI (apo AI) was determined after 4 h of incubation with macrophages.

Results

AA and DHA supplementation had no significant effect, whereas EPA induced a dose-dependent increase of ABCA1 functionality (+18% for 70 mM of EPA), without any alterations in ABCA1 expression. The EPA-treated macrophages exhibited strong phospholipid composition changes, with high levels of both EPA and its elongation product docosapentaenoic acid (DPA) (C22:5 n-3), which was associated with a decreased level of AA. Compared to control cells, EPA cells exhibited a higher capacity to bind apo A1. In addition, EPA membrane incorporation increased the ATPAse activity of ABCA1. We are currently investigating if EPA modulates the PKA pathway. We are also questioning whether EPA affects the cyclooxygenases (COX) activity and/or alters the balance of cellular eicosanoids produced from AA or EPA.

Conclusions

In conclusion, the in vitro EPA membrane incorporation increases ABCA1-mediated cholesterol efflux from THP-1 macrophages, which may partly explain its anti-atherogenic property confirmed in recent clinical trials.