Alirocumab, a monoclonal antibody that inhibits proprotein convertase subtilisin-kexin type 9 (PCSK9), has been shown to reduce low-density lipoprotein (LDL) cholesterol levels in patients who are ...receiving statin therapy. Larger and longer-term studies are needed to establish safety and efficacy.
We conducted a randomized trial involving 2341 patients at high risk for cardiovascular events who had LDL cholesterol levels of 70 mg per deciliter (1.8 mmol per liter) or more and were receiving treatment with statins at the maximum tolerated dose (the highest dose associated with an acceptable side-effect profile), with or without other lipid-lowering therapy. Patients were randomly assigned in a 2:1 ratio to receive alirocumab (150 mg) or placebo as a 1-ml subcutaneous injection every 2 weeks for 78 weeks. The primary efficacy end point was the percentage change in calculated LDL cholesterol level from baseline to week 24.
At week 24, the difference between the alirocumab and placebo groups in the mean percentage change from baseline in calculated LDL cholesterol level was -62 percentage points (P<0.001); the treatment effect remained consistent over a period of 78 weeks. The alirocumab group, as compared with the placebo group, had higher rates of injection-site reactions (5.9% vs. 4.2%), myalgia (5.4% vs. 2.9%), neurocognitive events (1.2% vs. 0.5%), and ophthalmologic events (2.9% vs. 1.9%). In a post hoc analysis, the rate of major adverse cardiovascular events (death from coronary heart disease, nonfatal myocardial infarction, fatal or nonfatal ischemic stroke, or unstable angina requiring hospitalization) was lower with alirocumab than with placebo (1.7% vs. 3.3%; hazard ratio, 0.52; 95% confidence interval, 0.31 to 0.90; nominal P=0.02).
Over a period of 78 weeks, alirocumab, when added to statin therapy at the maximum tolerated dose, significantly reduced LDL cholesterol levels. In a post hoc analysis, there was evidence of a reduction in the rate of cardiovascular events with alirocumab. (Funded by Sanofi and Regeneron Pharmaceuticals; ODYSSEY LONG TERM ClinicalTrials.gov number, NCT01507831.).
Obesity is a chronic disease with serious health consequences, but weight loss is difficult to maintain through lifestyle intervention alone. Liraglutide, a glucagon-like peptide-1 analogue, has been ...shown to have potential benefit for weight management at a once-daily dose of 3.0 mg, injected subcutaneously.
We conducted a 56-week, double-blind trial involving 3731 patients who did not have type 2 diabetes and who had a body-mass index (BMI; the weight in kilograms divided by the square of the height in meters) of at least 30 or a BMI of at least 27 if they had treated or untreated dyslipidemia or hypertension. We randomly assigned patients in a 2:1 ratio to receive once-daily subcutaneous injections of liraglutide at a dose of 3.0 mg (2487 patients) or placebo (1244 patients); both groups received counseling on lifestyle modification. The coprimary end points were the change in body weight and the proportions of patients losing at least 5% and more than 10% of their initial body weight.
At baseline, the mean (±SD) age of the patients was 45.1±12.0 years, the mean weight was 106.2±21.4 kg, and the mean BMI was 38.3±6.4; a total of 78.5% of the patients were women and 61.2% had prediabetes. At week 56, patients in the liraglutide group had lost a mean of 8.4±7.3 kg of body weight, and those in the placebo group had lost a mean of 2.8±6.5 kg (a difference of -5.6 kg; 95% confidence interval, -6.0 to -5.1; P<0.001, with last-observation-carried-forward imputation). A total of 63.2% of the patients in the liraglutide group as compared with 27.1% in the placebo group lost at least 5% of their body weight (P<0.001), and 33.1% and 10.6%, respectively, lost more than 10% of their body weight (P<0.001). The most frequently reported adverse events with liraglutide were mild or moderate nausea and diarrhea. Serious events occurred in 6.2% of the patients in the liraglutide group and in 5.0% of the patients in the placebo group.
In this study, 3.0 mg of liraglutide, as an adjunct to diet and exercise, was associated with reduced body weight and improved metabolic control. (Funded by Novo Nordisk; SCALE Obesity and Prediabetes NN8022-1839 ClinicalTrials.gov number, NCT01272219.).
Aim: Lipoprotein(a) (Lp(a)) is a low-density lipoprotein-like particle containing apolipoprotein(a) (apo(a)) that increases the risk of atherosclerotic cardiovascular disease (ASCVD) in familial ...hypercholesterolemia (FH). Postprandial redistribution of apo(a) protein from Lp(a) to triglyceride-rich lipoproteins (TRLs) may also increase the atherogenicity of TRL particles. Omega-3 fatty acid (ω3FA) supplementation improves postprandial TRL metabolism in FH subjects. However, its effect on postprandial apo(a) metabolism has yet to be investigated.Methods: We carried out an 8-week open-label, randomized, crossover trial to test the effect of ω3FA supplementation (4 g/day) on postprandial apo(a) responses in FH patients following ingestion of an oral fat load. Postprandial plasma total and TRL-apo(a) concentrations were measured by liquid chromatography with tandem mass spectrometry, and the corresponding areas under the curve (AUCs) (0–10h) were determined using the trapezium rule. Results: Compared with no ω3FA treatment, ω3FA supplementation significantly lowered the concentrations of postprandial TRL-apo(a) at 0.5 (−17.9%), 1 (−18.7%), 2 (−32.6%), and 3 h (−19.2%) (P＜0.05 for all). Postprandial TRL-apo(a) AUC was significantly reduced with ω3FA by 14.8% (P＜0.05). By contrast, ω3FA had no significant effect on the total AUCs of apo(a), apoC-III, and apoE (P＞0.05 for all). The decrease in postprandial TRL-apo(a) AUC was significantly associated with changes in the AUC of triglycerides (r=0.600; P＜0.01) and apoB-48 (r=0.616; P＜0.01).Conclusions: Supplementation with ω3FA reduces postprandial TRL-apo(a) response to a fat meal in FH patients; this novel metabolic effect of ω3FA may have implications on decreasing the risk of ASCVD in patients with FH, especially in those with elevated plasma triglyceride and Lp(a) concentrations. However, the clinical implications of these metabolic findings require further evaluation in outcome or surrogate endpoint trials.
Gut microbiota-dependent Trimethylamine-N-oxide (TMAO) has been reported to be strongly linked to renal function and to increased cardiovascular events in the general population and in Chronic Kidney ...Disease (CKD) patients. Considering the lack of data assessing renal handling of TMAO, we conducted this study to explore renal excretion and mechanisms of accumulation of TMAO during CKD. We prospectively measured glomerular filtration rate (mGFR) with gold standard methods and plasma concentrations of trimethylamine (TMA), TMAO, choline, betaine, and carnitine by LC-MS/MS in 124 controls, CKD, and hemodialysis (HD) patients. Renal clearance of each metabolite was assessed in a sub-group of 32 patients. Plasma TMAO was inversely correlated with mGFR (
= 0.388,
< 0.001), confirming elevation of TMAO plasma levels in CKD. TMAO clearances were not significantly different from mGFR, with a mean ± SD TMAO fractional excretion of 105% ± 32%. This suggests a complete renal excretion of TMAO by glomerular filtration with a negligible participation of tubular secretion or reabsorption, during all stages of CKD. Moreover, TMAO was effectively removed within 4 h of hemodiafiltration, showing a higher fractional reduction value than that of urea (84.9% ± 6.5% vs. 79.2% ± 5.7%,
= 0.04). This study reports a strong correlation between plasma TMAO levels and mGFR, in CKD, that can be mainly related to a decrease in TMAO glomerular filtration. Clearance data did not support a significant role for tubular secretion in TMAO renal elimination.
Human apoE exhibits three major isoforms (apoE2, apoE3, and apoE4) corresponding to polymorphism in the APOE gene. Total plasma apoE concentrations are closely related to these isoforms, but the ...underlying mechanisms are unknown. We aimed to describe the kinetics of apoE individual isoforms to explore the mechanisms for variable total apoE plasma concentrations. We used LC-MS/MS to discriminate between isoforms by identifying specific peptide sequences in subjects (three E2/E3, three E3/E3, and three E3/E4 phenotypes) who received a primed constant infusion of 2H3-leucine for 14 h. apoE concentrations and leucine enrichments were measured hourly in plasma. Concentrations of apoE2 were higher than apoE3, and concentrations of apoE4 were lower than apoE3. There was no difference between apoE3 and apoE4 catabolic rates and between apoE2 and apoE3 production rates (PRs), but apoE2 catabolic rates and apoE4 PRs were lower. The mechanisms leading to the difference in total plasma apoE concentrations are therefore related to contrasted kinetics of the isoforms. Production or catabolic rates are differently affected according to the specific isoforms. On these grounds, studies on the regulation of the involved biochemical pathways and the impact of pathological environments are now warranted.
Cardiovascular diseases are often associated with impaired lipid metabolism. Animal models are useful for deciphering the physiological mechanisms underlying these pathologies. However, lipid ...metabolism is contrasted between species limiting the transposition of findings from animals to human. Hence, we aimed to compare extended lipid profiles of several animal species to bring new insights in animal model selections. Human lipid phenotype was compared with those of 10 animal species. Standard plasma lipids and lipoprotein profiles were obtained by usual methods and lipidomic analysis was conducted by liquid chromatography-high-resolution mass spectrometry (LC-HRMS). As anticipated, we found contrasted lipid profiles between species. Some of them exhibited similar plasma lipids to human (non-human primate, rat, hamster, pig), but only usual lipid profiles of pigs were superimposable with human. LC-HRMS analyses allowed the identification of 106 other molecular species of lipids, common to all samples and belonging to major lipid families. Multivariate analyses clearly showed that hamster and, in a lower extent mouse, exhibited close lipid fingerprints to that of human. Besides, several lipid candidates that were previously reported to study cardiovascular diseases ranged similarly in human and hamster. Hence, hamster appeared to be the best option to study physiological disturbances related to cardiovascular diseases.
PCSK9 (Proprotein Convertase Subtilisin Kexin type 9) is a circulating protein that promotes hypercholesterolemia by decreasing hepatic LDL receptor protein. Under non interventional conditions, its ...expression is driven by sterol response element binding protein 2 (SREBP2) and follows a diurnal rhythm synchronous with cholesterol synthesis. Plasma PCSK9 is associated to LDL-C and to a lesser extent plasma triglycerides and insulin resistance. We aimed to verify the effect on plasma PCSK9 concentrations of dietary interventions that affect these parameters.
We performed nutritional interventions in young healthy male volunteers and offspring of type 2 diabetic (OffT2D) patients that are more prone to develop insulin resistance, including: i) acute post-prandial hyperlipidemic challenge (n=10), ii) 4 days of high-fat (HF) or high-fat/high-protein (HFHP) (n=10), iii) 7 (HFruc1, n=16) or 6 (HFruc2, n=9) days of hypercaloric high-fructose diets. An acute oral fat load was also performed in two patients bearing the R104C-V114A loss-of-function (LOF) PCSK9 mutation. Plasma PCSK9 concentrations were measured by ELISA. For the HFruc1 study, intrahepatocellular (IHCL) and intramyocellular lipids were measured by 1H magnetic resonance spectroscopy. Hepatic and whole-body insulin sensitivity was assessed with a two-step hyperinsulinemic-euglycemic clamp (0.3 and 1.0 mU.kg-1.min-1).
HF and HFHP short-term diets, as well as an acute hyperlipidemic oral load, did not significantly change PCSK9 concentrations. In addition, post-prandial plasma triglyceride excursion was not altered in two carriers of PCSK9 LOF mutation compared with non carriers. In contrast, hypercaloric 7-day HFruc1 diet increased plasma PCSK9 concentrations by 28% (p=0.05) in healthy volunteers and by 34% (p=0.001) in OffT2D patients. In another independent study, 6-day HFruc2 diet increased plasma PCSK9 levels by 93% (p<0.0001) in young healthy male volunteers. Spearman's correlations revealed that plasma PCSK9 concentrations upon 7-day HFruc1 diet were positively associated with plasma triglycerides (r=0.54, p=0.01) and IHCL (r=0.56, p=0.001), and inversely correlated with hepatic (r=0.54, p=0.014) and whole-body (r=-0.59, p=0.0065) insulin sensitivity.
Plasma PCSK9 concentrations vary minimally in response to a short term high-fat diet and they are not accompanied with changes in cholesterolemia upon high-fructose diet. Short-term high-fructose intake increased plasma PCSK9 levels, independent on cholesterol synthesis, suggesting a regulation independent of SREBP-2. Upon this diet, PCSK9 is associated with insulin resistance, hepatic steatosis and plasma triglycerides.
Familial autosomal dominant hypercholesterolemia is associated with high risk for cardiovascular accidents and is related to mutations in the low density lipoprotein receptor or its ligand ...apolipoprotein B (apoB). Mutations in a third gene, proprotein convertase subtilisin kexin 9 (PCSK9), were recently associated to this disease. PCSK9 acts as a natural inhibitor of the low density lipoprotein receptor pathway, and both genes are regulated by depletion of cholesterol cell content and statins, via sterol regulatory element-binding protein (SREBP). Here we investigated the regulation of PCSK9 gene expression during nutritional changes. We showed that PCSK9 mRNA quantity is decreased by 73% in mice after 24 h of fasting, leading to a 2-fold decrease in protein level. In contrast PCSK9 expression was restored upon high carbohydrate refeeding. PCSK9 mRNA increased by 4–5-fold in presence of insulin in rodent primary hepatocytes, whereas glucose had no effect. Moreover, insulin up-regulated hepatic PCSK9 expression in vivo during a hyperinsulinemic-euglycemic clamp in mice. Adenoviral mediated overexpression of a dominant or negative form of SREBP-1c confirmed the implication of this transcription factor in insulin-mediated stimulation of PCSK9 expression. Liver X receptor agonist T0901317 also regulated PCSK9 expression via this same pathway (a 2-fold increase in PCSK9 mRNA of primary hepatocytes cultured for 24 h in presence of 1 μm T0901317). As our last investigation, we isolated PCSK9 proximal promoter and verified the functionality of a SREBP-1c responsive element located from 335 bp to 355 bp upstream of the ATG. Together, these results show that PCSK9 expression is regulated by nutritional status and insulinemia.
Apolipoproteins govern lipoprotein metabolism and are promising biomarkers of metabolic and cardiovascular diseases. Unlike immunoassays, MS enables the quantification and phenotyping of multiple ...apolipoproteins. Hence, here, we aimed to develop a LC-MS/MS assay that can simultaneously quantitate 18 human apolipoproteins A-I, A-II, A-IV, A-V, B48, B100, C-I, C-II, C-III, C-IV, D, E, F, H, J, L1, M, and (a) and determined apoE, apoL1, and apo(a) phenotypes in human plasma and serum samples. The plasma and serum apolipoproteins were trypsin digested through an optimized procedure and peptides were extracted and analyzed by LC-MS/MS. The method was validated according to standard guidelines in samples spiked with known peptide amounts. The LC-MS/MS results were compared with those obtained with other techniques, and reproducibility, dilution effects, and stabilities were also assessed. Peptide markers were successfully selected for targeted apolipoprotein quantification and phenotyping. After optimization, the assay was validated for linearity, lower limits of quantification, accuracy (biases: –14.8% to 12.1%), intra-assay variability coefficients of variation (CVs): 1.5–14.2%, and inter-assay repeatability (CVs: 4.1–14.3%). Bland-Altman plots indicated no major statistically significant differences between LC-MS/MS and other techniques. The LC-MS/MS results were reproducible over five repeated experiments (CVs: 1.8–13.7%), and we identified marked differences among the plasma and serum samples. The LC-MS/MS assay developed here is rapid, requires only small sampling volumes, and incurs reasonable costs, thus making it amenable for a wide range of studies of apolipoprotein metabolism. We also highlight how this assay can be implemented in laboratories.
PDF
