Similar insulin regulation of splanchnic FFA and VLDL-TG in men with nonalcoholic hepatic steatosis and steatohepatitis.

This study aimed to determine whether obese men with nonalcoholic fatty liver disease (NAFLD) display differences between those with simple steatosis versus steatohepatitis (NASH) in splanchnic and hepatic FFA and VLDL-triglycerides (VLDL-TG) balances. The study involved 17 obese men with biopsy-proven NAFLD (9 with NASH and 8 with simple steatosis). We used hepatic vein catheterization in combination with [3H]palmitate and [14C]VLDL-TG tracers to measure splanchnic palmitate and VLDL-TG uptake and release rates during basal and hyperinsulinemic conditions. Indocyanine green was used to measure splanchnic plasma flow. Splanchnic palmitate uptake was similar in the two groups and significantly reduced during hyperinsulinemia (NASH: 62 (48-77) versus 38 (18-58) µmol/min; simple steatosis: 62 (46-78) versus 45 (25-65) µmol/min, mean (95% CI), basal versus clamp periods, respectively, P = 0.02 time-effect). Splanchnic palmitate release was also comparable between groups and nonsignificantly diminished during hyperinsulinemia. The percent palmitate delivered to the liver originating from visceral adipose tissue lipolysis was similar and unchanged by hyperinsulinemia. Splanchnic uptake and release of VLDL-TG were similar between groups. Hyperinsulinemia suppressed VLDL-TG release (P <0.05 time-effect) in both groups. Insulin-mediated glucose disposal was similar in the two groups (P = 0.54). Obese men with NASH and simple steatosis have similar splanchnic uptake and release of FFA and VLDL-TG and a similar proportion of FFA from visceral adipose tissue lipolysis delivered to the liver. These results demonstrate that the splanchnic balances of FFA and VLDL-TG do not differ between obese men with NASH and those with simple steatosis.

Acetyl-CoA carboxylase inhibitor increases LDL-apoB production rate in NASH with cirrhosis: prevention by fenofibrate.

Treatment with acetyl-CoA carboxylase inhibitors (ACCi) in nonalcoholic steatohepatitis (NASH) may increase plasma triglycerides (TGs), with variable changes in apoB concentrations. ACC is rate limiting in de novo lipogenesis and regulates fatty acid oxidation, making it an attractive therapeutic target in NASH. Our objectives were to determine the effects of the ACCi, firsocostat, on production rates of plasma LDL-apoB in NASH and the effects of combined therapy with fenofibrate. Metabolic labeling with heavy water and tandem mass spectrometric analysis of LDL-apoB enrichments was performed in 16 NASH patients treated with firsocostat for 12 weeks and in 29 NASH subjects treated with firsocostat and fenofibrate for 12 weeks. In NASH on firsocostat, plasma TG increased significantly by 17% from baseline to week 12 (P = 0.0056). Significant increases were also observed in LDL-apoB fractional replacement rate (baseline to week 12: 31 ± 20.2 to 46 ± 22.6%/day, P = 0.03) and absolute synthesis rate (ASR) (30.4-45.2 mg/dl/day, P = 0.016) but not plasma apoB concentrations. The effect of firsocostat on LDL-apoB ASR was restricted to patients with cirrhosis (21.0 ± 9.6 at baseline and 44.2 ± 17 mg/dl/day at week 12, P = 0.002, N = 8); noncirrhotic patients did not change (39.8 ± 20.8 and 46.3 ± 14.8 mg/dl/day, respectively, P = 0.51, N = 8). Combination treatment with fenofibrate and firsocostat prevented increases in plasma TG, LDL-apoB fractional replacement rate, and ASR. In summary, in NASH with cirrhosis, ACCi treatment increases LDL-apoB100 production rate and this effect can be prevented by concurrent fenofibrate therapy.

A mathematical model to estimate cholesterylester transfer protein (CETP) triglycerides flux in human plasma.

BACKGROUND: Cholesterylester transfer protein (CETP) modulates the composition of various lipoproteins associated with cardiovascular disease. Despite its central role in lipoprotein metabolism, its mode of action is still not fully understood. Here we present a simple way to estimate CETP-mediated lipid fluxes between different lipoprotein fractions. RESULTS: The model derived adequately describes the observed findings, especially regarding low- and high dense lipoproteins (LDL and HDL), delivering correlation coefficients of R2 = 0.567 (p < 0.001) and R2 = 0.466 (p < 0.001), respectively. These estimated fluxes correlate best among all other measured concentrations and 'lipid per lipoprotein' ratios to the observed fluxes. CONCLUSION: Our model approach is independent of CETP-action's exact mechanistic mode. It is simple and easy to apply, and may be a useful tool in revealing CETP's ambiguous role in lipid metabolism. The model mirrors a diffusion-like exchange of triglycerides between lipoproteins. Cholesteryl ester and triglyceride concentrations measured in HDL, LDL and VLDL are sufficient to apply the model on a plasma sample.

The metabolism of lipoprotein (a): an ever-evolving story.

Lipoprotein (a) [Lp(a)] is characterized by apolipoprotein (a) [apo(a)] covalently bound to apolipoprotein B 100. It was described in human plasma by Berg et al. in 1963 and the gene encoding apo(a) (LPA) was cloned in 1987 by Lawn and colleagues. Epidemiologic and genetic studies demonstrate that increases in Lp(a) plasma levels increase the risk of atherosclerotic cardiovascular disease. Novel Lp(a) lowering treatments highlight the need to understand the regulation of plasma levels of this atherogenic lipoprotein. Despite years of research, significant uncertainty remains about the assembly, secretion, and clearance of Lp(a). Specifically, there is ongoing controversy about where apo(a) and apoB-100 bind to form Lp(a); which apoB-100 lipoproteins bind to apo(a) to create Lp(a); whether binding of apo(a) is reversible, allowing apo(a) to bind to more than one apoB-100 lipoprotein during its lifespan in the circulation; and how Lp(a) or apo(a) leave the circulation. In this review, we highlight past and recent data from stable isotope studies of Lp(a) metabolism, highlighting the critical metabolic uncertainties that exist. We present kinetic models to describe results of published studies using stable isotopes and suggest what future studies are required to improve our understanding of Lp(a) metabolism.

Metabolism and proteomics of large and small dense LDL in combined hyperlipidemia: effects of rosuvastatin.

Small dense LDL (sdLDL) has been reported to be more atherogenic than large buoyant LDL (lbLDL). We examined the metabolism and protein composition of sdLDL and lbLDL in six subjects with combined hyperlipidemia on placebo and rosuvastatin 40 mg/day. ApoB-100 kinetics in triglyceride-rich lipoproteins (TRLs), lbLDL (density [d] = 1.019-1.044 g/ml), and sdLDL (d = 1.044-1.063 g/ml) were determined in the fed state by using stable isotope tracers, mass spectrometry, and compartmental modeling. Compared with placebo, rosuvastatin decreased LDL cholesterol and apoB-100 levels in TRL, lbLDL, and sdLDL by significantly increasing the fractional catabolic rate of apoB-100 (TRL, +45%; lbLDL, +131%; and sdLDL, +97%), without a change in production. On placebo, 25% of TRL apoB-100 was catabolized directly, 37% was converted to lbLDL, and 38% went directly to sdLDL; rosuvastatin did not alter these distributions. During both phases, sdLDL apoB-100 was catabolized more slowly than lbLDL apoB-100 (P < 0.01). Proteomic analysis indicated that rosuvastatin decreased apoC-III and apoM content within the density range of lbLDL (P < 0.05). In our view, sdLDL is more atherogenic than lbLDL because of its longer plasma residence time, potentially resulting in more particle oxidation, modification, and reduction in size, with increased arterial wall uptake. Rosuvastatin enhances the catabolism of apoB-100 in both lbLDL and sdLDL.

An in silico model of retinal cholesterol dynamics (RCD model): insights into the pathophysiology of dry AMD.

We developed an in silico mathematical model of retinal cholesterol (Ch) dynamics (RCD) to quantify the physiological rate of Ch turnover in the rod outer segment (ROS), the lipoprotein transport mechanisms by which Ch enters and leaves the outer retina, and the rates of drusen growth and macrophage-mediated clearance in dry age-related macular degeneration. Based on existing experimental data and mechanistic hypotheses, we estimated the Ch turnover rate in the ROS to be 1-6 pg/mm2/min, dependent on the rate of Ch recycling in the outer retina, and found comparable rates for LDL receptor-mediated endocytosis of Ch by the retinal pigment epithelium (RPE), ABCA1-mediated Ch transport from the RPE to the outer retina, ABCA1-mediated Ch efflux from the RPE to the choroid, and the secretion of 70 nm ApoB-Ch particles from the RPE. The drusen growth rate is predicted to increase from 0.7 to 4.2 µm/year in proportion to the flux of ApoB-Ch particles. The rapid regression of drusen may be explained by macrophage-mediated clearance if the macrophage density reaches ∼3,500 cells/mm2 The RCD model quantifies retinal Ch dynamics and suggests that retinal Ch turnover and recycling, ApoB-Ch particle efflux, and macrophage-mediated clearance may explain the dynamics of drusen growth and regression.

In silico modeling of the dynamics of low density lipoprotein composition via a single plasma sample.

Lipoproteins play a key role in the development of CVD, but the dynamics of lipoprotein metabolism are difficult to address experimentally. This article describes a novel two-step combined in vitro and in silico approach that enables the estimation of key reactions in lipoprotein metabolism using just one blood sample. Lipoproteins were isolated by ultracentrifugation from fasting plasma stored at 4°C. Plasma incubated at 37°C is no longer in a steady state, and changes in composition may be determined. From these changes, we estimated rates for reactions like LCAT (56.3 µM/h), ß-LCAT (15.62 µM/h), and cholesteryl ester (CE) transfer protein-mediated flux of CE from HDL to IDL/VLDL (21.5 µM/h) based on data from 15 healthy individuals. In a second step, we estimated LDL's HL activity (3.19 pools/day) and, for the very first time, selective CE efflux from LDL (8.39 µM/h) by relying on the previously derived reaction rates. The estimated metabolic rates were then confirmed in an independent group (n = 10). Although measurement uncertainties do not permit us to estimate parameters in individuals, the novel approach we describe here offers the unique possibility to investigate lipoprotein dynamics in various diseases like atherosclerosis or diabetes.

Multiplexed peptide analysis for kinetic measurements of major human apolipoproteins by LC/MS/MS.

A multiplexed assay was developed by MS to analyze, in a single run, six major human Apos involved in lipoprotein metabolism: ApoA-I, ApoA-II, ApoB100, ApoC-II, ApoC-III, and ApoE. This method was validated in vivo in six subjects who received a 14 h constant infusion of [5,5,5-(2)H3]L-leucine at 10 µM/kg/h. Plasma lipoprotein fractions were isolated from collected blood samples and were digested with trypsin. Proteotypic peptides were subsequently analyzed by LC/MS/MS. Enrichment measurement data were compared with those obtained by the standard method using GC/MS. The required time to obtain the LC/MS/MS data was less than that needed for GC/MS. The enrichments from both methods were correlated for ApoA-I (r = 0.994; P < 0.0001) and ApoB100 (r = 0.999; P < 0.0001), and the Bland-Altman plot confirmed the similarity of the two methods. Intra- and inter-assay variability calculated for the six Apos of interest did not exceed 10.7 and 12.5%, respectively, and kinetic parameters were similar and/or in agreement with previously reported data. Therefore, LC/MS/MS can be considered as a useful tool for human Apo kinetic studies using stable isotopes.

Lipoprotein (a): truly a direct prothrombotic factor in cardiovascular disease?

Elevated plasma concentrations of lipoprotein (a) [Lp(a)] have been determined to be a causal risk factor for coronary heart disease, and may similarly play a role in other atherothrombotic disorders. Lp(a) consists of a lipoprotein moiety indistinguishable from LDL, as well as the plasminogen-related glycoprotein, apo(a). Therefore, the pathogenic role for Lp(a) has traditionally been considered to reflect a dual function of its similarity to LDL, causing atherosclerosis, and its similarity to plasminogen, causing thrombosis through inhibition of fibrinolysis. This postulate remains highly speculative, however, because it has been difficult to separate the prothrombotic/antifibrinolytic functions of Lp(a) from its proatherosclerotic functions. This review surveys the current landscape surrounding these issues: the biochemical basis for procoagulant and antifibrinolytic effects of Lp(a) is summarized and the evidence addressing the role of Lp(a) in both arterial and venous thrombosis is discussed. While elevated Lp(a) appears to be primarily predisposing to thrombotic events in the arterial tree, the fact that most of these are precipitated by underlying atherosclerosis continues to confound our understanding of the true pathogenic roles of Lp(a) and, therefore, the most appropriate therapeutic target through which to mitigate the harmful effects of this lipoprotein.

Excessive centrifugal fields damage high density lipoprotein.

HDL is typically isolated ultracentrifugally at 40,000 rpm or greater, however, such high centrifugal forces are responsible for altering the recovered HDL particle. We demonstrate that this damage to HDL begins at approximately 30,000 rpm and the magnitude of loss increases in a rotor speed-dependent manner. The HDL is affected by elevated ultracentrifugal fields resulting in a lower particle density due to the shedding of associated proteins. To circumvent the alteration of the recovered HDL, we utilize a KBr-containing density gradient and a lowered rotor speed of 15,000 rpm to separate the lipoproteins using a single 96 h centrifugation step. This recovers the HDL at two density ranges; the bulk of the material has a density of about 1.115 g/ml, while lessor amounts of material are recovered at >1.2 g/ml. Thus, demonstrating the isolation of intact HDL is possible utilizing lower centrifuge rotor speeds.