Fish Oil Supplementation Modifies the Proteome, Lipidome, and Function of High-Density Lipoprotein: Findings from a Trial in Young Healthy Adults.

BACKGROUND: Fish oil with the ω-3 fatty acids EPA and DHA is an FDA-approved treatment of patients with severe hypertriglyceridemia. Furthermore, EPA is an FDA-approved treatment of patients with high risk of cardiovascular disease (CVD); however, the cardioprotective mechanisms are unclear. OBJECTIVES: We aimed to determine if fish oil supplementation is cardioprotective due to beneficial modifications in HDL particles. METHODS: Seven fish oil naïve subjects without a history of CVD were recruited to take a regimen of fish oil (1125 mg EPA and 875 mg DHA daily) for 30 d, followed by a 30-d washout period wherein no fish oil supplements were taken. HDL isolated from fasting whole blood at each time point via 2-step ultracentrifugation (ucHDL) was assessed for proteome, lipidome, cholesterol efflux capacity (CEC), and anti-inflammatory capacity. RESULTS: Following fish oil supplementation, the HDL-associated proteins immunoglobulin heavy constant γ1, immunoglobulin heavy constant α1, apolipoprotein D, and phospholipid transfer protein decreased compared to baseline (P < 0.05). The HDL-associated phospholipid families sphingomyelins, phosphatidylcholines, and phosphatidylserines increased after fish oil supplementation relative to baseline (P < 0.05). Compared to baseline, fish oil supplementation increased serum HDL's CEC (P = 0.002). Fish oil-induced changes (Post compared with Baseline) in serum HDL's CEC positively correlated with plasma EPA levels (R2 = 0.7256; P = 0.015). Similarly, fish oil-induced changes in ucHDL's CEC positively correlated with ucHDL's ability to reduce interleukin 10 (R2 = 0.7353; P = 0.014) and interleukin 6 mRNA expression (R2 = 0.6322; P =0.033) in a human macrophage cell line. CONCLUSIONS: Overall, fish oil supplementation improved HDL's sterol efflux capacity through comprehensive modifications to its proteome and lipidome.

High-Density Lipoprotein Carries Markers That Track With Recovery From Stroke.

RATIONALE: Prospective cohort studies question the value of HDL-C (high-density lipoprotein cholesterol) for stroke risk prediction. OBJECTIVE: Investigate the relationship between long-term functional recovery and HDL proteome and function. METHODS AND RESULTS: Changes in HDL protein composition and function (cholesterol efflux capacity) in patients after acute ischemic stroke at 2 time points (24 hours, 35 patients; 96 hours, 20 patients) and in 35 control subjects were measured. The recovery from stroke was assessed by 3 months, the National Institutes of Health Stroke Scale and modified Rankin scale scores. When compared with control subject after adjustments for sex and HDL-C levels, 12 proteins some of which participate in acute phase response and platelet activation (APMAP [adipocyte plasma membrane-associated protein], GPLD1 [phosphate inositol-glycan specific phospholipase D], APOE [apolipoprotein E], IHH [Indian hedgehog protein], ITIH4 [inter-alpha-trypsin inhibitor chain H4], SAA2 [serum amyloid A2], APOA4 [apolipoprotein A-IV], CLU [clusterin], ANTRX2 [anthrax toxin receptor 2], PON1 [serum paraoxonase/arylesterase], SERPINA1 [alpha-1-antitrypsin], and APOF [apolipoprotein F]) were significantly (adjusted P<0.05) altered in stroke HDL at 96 hours. The first 8 of these proteins were also significantly altered at 24 hours. Consistent with inflammatory remodeling, cholesterol efflux capacity was reduced by 32% (P<0.001) at both time points. Baseline stroke severity adjusted regression model showed that changes within 96-hour poststroke in APOF, APOL1, APMAP, APOC4 (apolipoprotein C4), APOM (apolipoprotein M), PCYOX1 (prenylcysteine oxidase 1), PON1, and APOE correlate with stroke recovery scores (R2=0.38-0.73, adjusted P<0.05). APOF (R2=0.73) and APOL1 (R2=0.60) continued to significantly correlate with recovery scores after accounting for tPA (tissue-type plasminogen activator) treatment. CONCLUSIONS: Changes in HDL proteins during early acute phase of stroke associate with recovery. Monitoring HDL proteins may provide clinical biomarkers that inform on stroke recuperation.

A method for lipoprotein (a) Isolation from a small volume of plasma with applications for clinical research.

High levels of circulating Lipoprotein (a) [Lp(a)] are an independent risk factor for CVD. One of the major limitations to investigating Lp(a) biology is the need for large volumes of plasma (4-10 mL) for its isolation. We developed an isolation technique requiring only 0.4 mL of plasma yielding an enriched Lp(a) fraction suitable for compositional and functional studies. We collected plasma from patients (n = 9) in EDTA presenting to our Center for Preventive Cardiology for CVD risk management and with circulating Lp(a) > 66 mg/dL. 0.4 mL of plasma was added to 90 µL of potassium bromide (1.33 g/mL) and subjected to our two-step density-gradient ultracentrifugation method. The first step separates VLDL and LDL from the Lp(a) and HDL fractions and the second step further separates VLDL from LDL and Lp(a) from HDL. Lp(a) is then dialyzed for up to 24 h in potassium phosphate buffer. We performed cholesterol gel electrophoresis, immunoblotting and LC-MS/MS proteomics on isolated lipoprotein fractions to confirm fraction enrichment. Functional studies including Lp(a)-dependent induction of macrophage gene expression and cholesterol efflux inhibition were performed on isolated Lp(a) to confirm its preserved bioactivity. Lp(a) yields (264 ± 82.3 µg/mL on average) correlated with Lp(a) plasma concentrations (r2 = 0.75; p < 0.01) and represented the relative distribution of circulating apo(a) isoforms. Proteomic analyses confirm lipoprotein fraction separation. Functional integrity was confirmed by the findings that isolated Lp(a) inhibited plasminogen-dependent cholesterol efflux in HEK293T cells expressing ABCA1 and increased expressions of Il1b, Nos2 and Ccl2. We developed a small-volume isolation technique for Lp(a) suited for a range of applications used in biomedical research. The use of this technique circumvents volume-dependent limitations and expands our ability to investigate the mysteries of this deleterious lipoprotein.

Elevated Lipoprotein(a) Levels Lower ABCA1 Cholesterol Efflux Capacity.

CONTEXT: Elevated serum lipoprotein(a) [Lp(a)] levels are associated with increased cardiovascular disease risk. ABCA1-mediated cholesterol efflux from macrophages may be an antiatherogenic process. Plasminogen (PLG) is a driver of ABCA1-mediated cholesterol efflux, and its action is inhibited by purified human Lp(a). OBJECTIVE: To determine the effects of Lp(a) in human serum on ABCA1 cholesterol efflux. METHODS: Cholesterol efflux capacity (CEC) was measured with two different cell-culture models using serum from 76 patients with either low (<50 mg/dL) or high (>50 mg/dL) Lp(a) levels. RESULTS: Using cAMP-stimulated J774 macrophages or baby hamster kidney fibroblasts overexpressing human ABCA1, we show that CEC was lower in patients with high Lp(a) levels compared with patients with low levels (-30.6%, P = 0.002 vs -24.1%, P < 0.001, respectively). Total-serum CEC negatively correlated with Lp(a) levels (r = -0.433, P = 0.0007 vs r = -0.505, P = 0.0011, respectively). These negative associations persisted after adjusting for serum cholesterol, age, sex, and statin use in a multiple linear regression model (adjusted R2 = 0.413 or 0.405, respectively) and were strengthened when further adjusting for the interaction between Lp(a) and PLG levels (adjusted R2 = 0.465 and 0.409, respectively). Total-serum and isolated Lp(a) from patients with high Lp(a) inhibited PLG-mediated ABCA1 cholesterol efflux. CONCLUSION: Total-serum CEC is reduced in patients with high Lp(a) levels. This is in part due to the inhibition of PLG-mediated ABCA1 cholesterol efflux by Lp(a). Our findings suggest an atherogenic role for Lp(a) through its ability to inhibit CEC.

GM-CSF driven myeloid cells in adipose tissue link weight gain and insulin resistance via formation of 2-aminoadipate.

In a GM-CSF driven myeloid cell deficient mouse model (Csf2-/-) that has preserved insulin sensitivity despite increased adiposity, we used unbiased three-dimensional integration of proteome profiles, metabolic profiles, and gene regulatory networks to understand adipose tissue proteome-wide changes and their metabolic implications. Multi-dimensional liquid chromatography mass spectrometry and extended multiplex mass labeling was used to analyze proteomes of epididymal adipose tissues isolated from Csf2+/+ and Csf2-/- mice that were fed low fat, high fat, or high fat plus cholesterol diets for 8 weeks. The metabolic health (as measured by body weight, adiposity, plasma fasting glucose, insulin, triglycerides, phospholipids, total cholesterol levels, and glucose and insulin tolerance tests) deteriorated with diet for both genotypes, while mice lacking Csf2 were protected from insulin resistance. Regardless of diet, 30 mostly mitochondrial, branch chain amino acids (BCAA), and lysine metabolism proteins were altered between Csf2-/- and Csf2+/+ mice (FDR < 0.05). Lack of GM-CSF driven myeloid cells lead to reduced adipose tissue 2-oxoglutarate dehydrogenase complex (DHTKD1) levels and subsequent increase in plasma 2-aminoadipate (2-AA) levels, both of which are reported to correlate with insulin resistance. Tissue DHTKD1 levels were >4-fold upregulated and plasma 2-AA levels were >2 fold reduced in Csf2-/- mice (p < 0.05). GM-CSF driven myeloid cells link peripheral insulin sensitivity to adiposity via lysine metabolism involving DHTKD1/2-AA axis in a diet independent manner.