Plasma Lipidome Analysis by Liquid Chromatography-High Resolution Mass Spectrometry and Ion Mobility of Hypertriglyceridemic Patients on Extended-Release Nicotinic Acid: a Pilot Study.

BACKGROUND: Plasma high triacylglycerols and low HDL-C concentration are associated with increased cardiovascular events. Extended-release nicotinic acid (ERN) was shown to reduce plasma triacylglycerols and total cholesterol but also to markedly increase high-density lipoprotein-cholesterol (HDL-C). No data on the effect of ERN on different species of triacylglycerol, cholesteryl ester, and phospholipids are available. In this study, we applied a nontargeted lipidomic approach to investigate the plasma and lipoproteins lipids profile of hypertriglyceridemic patients treated with ERN or a placebo in order to identify new lipids markers associated with this treatment. METHODS: Eight hypertriglyceridemic patients enrolled in a crossover randomized trial with ERN for 8 weeks and treated with 2 g/day of ERN or a placebo. Ultra-performance liquid chromatography (UPLC) coupled to high-resolution mass spectrometry (HRMS) was used in mass spectrometry energy mode (HRMSE) combined with ion mobility spectrometry to characterize the plasma and very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) lipidome. The accuracy and precision of the method were validated on plasma samples. RESULTS: Compared to placebo, among 155 plasma lipids characterized using UPLC-ESI-HRMS, a multivariate analysis revealed a significant increase of lysophosphatidylcholine (LPC 20:5), a significant decrease of phosphatidylethanolamine (PE 16:0/22:3) and sphingomyelin (SM d18:1/22:0) and a decrease of triacylglycerol (TG 16:0/16:1/18:2) after ERN treatment. Analysis of these lipids in lipoproteins showed an increase of LPC (20:5) in HDL, a decrease of PE (16:0/22:3) in HDL and LDL, of SM (d18:1/22:0) in VLDL and LDL and of TG (16:0/16:1/18:2) in VLDL. CONCLUSION: This lipidomic strategy characterized new specific lipid markers likely to be involved in the effect of ERN on cardiovascular risk opening a new strategy to analyze randomized controlled with this treatment. TRIAL REGISTRATION: NCT01216956.

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.

Low rate of production of apolipoproteins B100 and AI in 2 patients with Anderson disease (chylomicron retention disease).

OBJECTIVE: Anderson disease is a rare inherited lipid malabsorption syndrome associated with hypocholesterolemia and linked to SAR1B mutations. The aim of this article was to analyze the mechanisms responsible for the low plasma apolipoprotein Apo-B100 and Apo-AI in 2 patients with Anderson disease. METHODS AND RESULTS: A primed constant infusion of (13)C-leucine was administered for 14 hours to determine the kinetics of lipoproteins. In the 2 patients, total cholesterol (77 and 85 mg/dL versus 155±32 mg/dL), triglycerides (36 and 59 versus 82±24 mg/dL), Apo-B100 (48 and 43 versus 71±5 mg/dL), and Apo-AI (47 and 62 versus 130±7 mg/dL) were lower compared with 6 healthy individuals. Very-low-density lipoprotein-B100 production rate of the patients was lower (4.08 and 5.52 mg/kg/day versus 12.96±2.88 mg/kg/day) as was the fractional catabolic rate (5.04 and 4.32 day(-1) versus 12.24±3.84 day(-1)). No difference was observed in intermediate-density lipoprotein-B100 and LDL-B100 kinetic data. The production rate of high-density lipoprotein Apo-AI was lower in the patients (7.92 and 8.64 versus 11.96±1.92 mg/kg/day) and the fractional catabolic rate was higher (0.38 and 0.29 versus 0.22±0.01 day(-1)). CONCLUSIONS: The low plasma Apo-B100 and Apo-AI concentrations in the patients with Anderson disease were mainly related to low rates of production.

Effect on LDL-cholesterol of a large dose of a dietary supplement with plant extracts in subjects with untreated moderate hypercholesterolaemia: a randomised, double-blind, placebo-controlled study.

PURPOSE: To determine the effect of 4 weeks of supplementation, then, withdrawal of a dietary supplement (DS) containing red yeast rice extract, policosanol and artichoke leaf extract at twice the recommended daily dose (6 tablets, 6-TAB) compared to the usual dose (3-TAB) or to a placebo (PLA), on blood lipid profiles and safety biomarkers. METHODS: Forty-five healthy subjects (15 per group), with untreated hypercholesterolaemia, were included in this randomised, double-blind, placebo-controlled clinical trial. RESULTS: After 4 weeks of supplementation, LDL-C was significantly lower in 6-TAB (-0.21 g/l; 95 % CI -0.38 to -0.03 g/l; p = 0.0217) and 3-TAB (-0.25 g/l; 95 % CI -0.42 to -0.07 g/l; p = 0.0071) compared to PLA, although no difference in LDL-cholesterol was observed between the two groups, while no effect was seen on triacylglycerol and HDL-cholesterol. Four weeks after the end of supplementation, no difference in LDL-C was seen between the PLA group and the DS-treated groups. The muscle breakdown biomarkers, as well as biomarkers of liver and renal function, were altered by neither dose of the DS. Acute application of the DS on permeabilised skeletal muscle fibres of rats did not induce deleterious effects on mitochondrial function. CONCLUSIONS: Supplementation with twice the recommended dose of the DS was effective in reducing LDL-cholesterol and appeared safe, but according to the present results, no additional benefit could be achieved compared to the recommended dose.

Effect of a high bicarbonate mineral water on fasting and postprandial lipemia in moderately hypercholesterolemic subjects: a pilot study.

BACKGROUND: During postprandial state, TG concentration is increasing and HDL cholesterol decreasing, leading to a transitory pro-atherosclerotic profile. Previous studies have reported that bicarbonate water improve postprandial lipemia. The objective of this study was to analyze the effect of a strongly bicarbonated mineral water on lipoprotein levels during fasting and postprandial state. METHODS: A controlled, randomised, double-blind cross-over design was conducted in 12 moderately hypercholesterolemic subjects after a daily ingestion of 1.25 L of mineral (SY) or low mineral water during eight weeks separated by a one week wash-out period. Blood samples were collected in first visit to the hospital (V1) before water consumption (referent or SY) and in a second visit (V2) after eight week water consumption period. The effect of the consumed water was studied in fasting and in postprandial state during ingestion of a meal and 0.5 L of water. RESULTS: Comparison of data between V1 and V2 after SY consumption showed a significant decrease in triglyceridemia (23%), VLDL TG (31%) and tendency to a decrease of VLDL cholesterol (p = 0.066) at fasting state. Whatever the consumed water during postprandial state, the measurement of total areas under curves did not show a significant difference. No difference was observed between SY and referent water consumption for measured parameters at fasting and postprandial state. CONCLUSION: When subjects consumed SY we showed a decrease of their basal TG and VLDLTG. The unexpected absence of effect of high mineralized water on postprandial lipemia, probably related to experimental conditions, is discussed in the discussion section.

A combined natural supplement lowers LDL cholesterol in subjects with moderate untreated hypercholesterolemia: a randomized placebo-controlled trial.

OBJECTIVE: To investigate the effect of a natural cholesterol-lowering supplement (NCLS) containing red yeast rice, policosanols and artichoke leaf extracts on blood lipid concentrations as well as on safety parameters when given over 16 weeks in 100 volunteers with untreated moderate hypercholesterolemia, in a randomized, double-blind, placebo-controlled trial. RESULTS: Reduction of primary outcome low-density lipoprotein cholesterol [-0.22 g/L (95% confidence interval, CI: -0.31 to -0.12) corresponding to -14.3% from baseline (95% CI: -21.5 to -7.2) compared to placebo], as well as total cholesterol, apolipoprotein B100 and apolipoprotein B100/apolipoprotein A-I ratio, were observed after 16 weeks of supplementation with NCLS. These effects were already observed at Week 4 and 10 of supplementation. No significant changes were observed in high-density lipoprotein, triacylglycerol, creatine kinase, lactate dehydrogenase and coenzyme Q10 levels, as well as in markers of liver and renal function. CONCLUSIONS: The NCLS was effective in reducing low-density lipoprotein cholesterol and apolipoprotein B100 in subjects with moderate hypercholesterolemia, without modifying safety parameters.

PCSK9 dominant negative mutant results in increased LDL catabolic rate and familial hypobetalipoproteinemia.

OBJECTIVE: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a central player in the regulation of cholesterol homeostasis, increasing the low-density lipoprotein (LDL) receptor degradation. Our study aimed at exploring the pathogenic consequences in vivo and in vitro of a PCSK9 prodomain mutation found in a family with hypobetalipoproteinemia (FHBL). METHODS AND RESULTS: A white 49-year-old diabetic man had profound FBHL (LDLC: 16 mg/dL) whereas his daughter and sister displayed a milder phenotype (LDLC 44 mg/dL and 57 mg/dL, respectively), all otherwise healthy with a normal liver function. A monoallelic PCSK9 double-mutant R104C/V114A cosegregated with FBHL, with no mutation found at other FHBL-causing loci. A dose-effect was also found in FBHL relatives for plasma APOB and PCSK9 (very-low to undetectable in proband, approximately 50% decreased in sister and daughter) and LDL catabolic rate (256% and 88% increased in proband and daughter). Transient transfection in hepatocytes showed severely impaired processing and secretion of the double mutant which acted as a dominant negative over secretion of wild-type PCSK9. CONCLUSIONS: These results show that heterozygous PCSK9 missense mutations may associate with profound hypobetalipoproteinemia and constitute the first direct evidence in human that decrease of plasma LDLC concentrations associated to PCSK9 LOF mutations are attributable to an increased clearance rate of LDL.

Omega 3 Improves Both apoB100-containing Lipoprotein Turnover and their Sphingolipid Profile in Hypertriglyceridemia.

CONTEXT: Evidence for an association between sphingolipids and metabolic disorders is increasingly reported. Omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFAs) improve apolipoprotein B100 (apoB100)-containing lipoprotein metabolism, but their effects on the sphingolipid content in lipoproteins remain unknown. OBJECTIVES: In subjects with hypertriglyceridemia, we analyzed the effect of n-3 LC-PUFAs on the turnover apoB100-containing lipoproteins and on their sphingolipid content and looked for the possible association between these lipid levels and apoB100-containing lipoprotein turnover parameters. METHODS: Six subjects underwent a kinetic study before and after n-3 supplementation for 2 months with 1 g of fish oil 3 times day containing 360 mg of eicosapentaenoic acid (EPA) and 240 mg of docosahexaenoic acid (DHA) in the form of triglycerides. We examined apoB100-containing lipoprotein turnover by primed perfusion labeled [5,5,5-2H3]-leucine and determined kinetic parameters using a multicompartmental model. We quantified sphingolipid species content in lipoproteins using mass spectrometry. RESULTS: Supplementation decreased very low-density lipoprotein (VLDL), triglyceride, and apoB100 concentrations. The VLDL neutral and polar lipids showed increased n-3 LC-PUFA and decreased n-6 LC-PUFA content. The conversion rate of VLDL1 to VLDL2 and of VLDL2 to LDL was increased. We measured a decrease in total apoB100 production and VLDL1 production. Supplementation reduced the total ceramide concentration in VLDL while the sphingomyelin content in LDL was increased. We found positive correlations between plasma palmitic acid and VLDL ceramide and between VLDL triglyceride and VLDL ceramide, and inverse correlations between VLDL n-3 LC-PUFA and VLDL production. CONCLUSION: Based on these results, we hypothesize that the improvement in apoB100 metabolism during n-3 LC-PUFA supplementation is contributed to by changes in sphingolipids.

Effect of fasting and feeding on apolipoprotein A-I kinetics in preß1-HDL, α-HDL, and triglyceride-rich lipoproteins.

The aim of this study was to compare the kinetics of apolipoprotein (apo)A-I during fed and fasted states in humans, and to determine to what extent the intestine contributes to apoA-I production. A stable isotope study was conducted to determine the kinetics of apoA-I in preß1 high-density lipoprotein (HDL) and α-HDL. Six healthy male subjects received a constant intravenous infusion of 2H3-leucine for 14 h. Subjects in the fed group also received small hourly meals. Blood samples were collected hourly during tracer infusion and then daily for 4 days. Tracer enrichments were measured by mass spectrometry and then fitted to a compartmental model using asymptotic plateau of very-low-density lipoprotein (VLDL) apoB100 and triglyceride-rich lipoprotein (TRL) apoB48 as estimates of hepatic and intestinal precursor pools, respectively. The clearance rate of preß1-HDL-apoA-I was lower in fed individuals compared with fasted subjects (p < 0.05). No other differences in apoA-I production or clearance rates were observed between the groups. No significant correlation was observed between plasma apoC-III concentrations and apoA-I kinetic data. In contrast, HDL-apoC-III was inversely correlated with the conversion of α-HDL to preß1-HDL. Total apoA-I synthesis was not significantly increased in fed subjects. Hepatic production was not significantly different between the fed group (17.17 ± 2.75 mg/kg/day) and the fasted group (18.67 ± 1.69 mg/kg/day). Increase in intestinal apoA-I secretion in fed subjects was 2.20 ± 0.61 mg/kg/day. The HDL-apoA-I kinetics were similar in the fasted and fed groups, with 13% of the total apoA-I originating from the intestine with feeding.

Fatty liver index is a strong predictor of changes in glycemic status in people with prediabetes: The IT-DIAB study.

BACKGROUND & AIMS: In patients at metabolic risk, nonalcoholic fatty liver disease is a strong and highly prevalent predictor for type 2 diabetes. Its assessment in clinical practice is not easy but the fatty liver index (FLI) could be used as a surrogate. Here, we studied the association between the FLI and the conversion to new-onset diabetes (NOD) or prediabetes reversion in patients with prediabetes. METHODS: The IT-DIAB observational study included 389 individuals with prediabetes, defined as fasting plasma glucose (FPG) between 110 and 125 mg/dL. NOD conversion was defined as a first FPG value ≥ 126 mg/dL and prediabetes reversion as a first FPG value < 110 mg/dL. The associations of both events with baseline FLI were studied separately using multivariate Cox models. RESULTS: After a median follow-up of 3.9 years (range 0.1-6.1), 138 individuals (35.5%) converted to NOD. FLI was associated with a higher risk of NOD conversion (unadjusted HR per SD = 1.54, 95%CI 1.27-1.86, p<0.0001), even after multiple adjustment on FPG, HbA1c and diabetes risk score (adjusted HR per SD 1.31, 95%CI 1.07-1.61, p = 0.008). FLI was also associated with prediabetes reversion: adjusted HR per SD = 0.85, 95%CI 0.75-0.96, p = 0.0077. Changes in FLI were significantly associated with changes in FPG during follow-up (p<0.0001). When compared to a full model including the diabetes risk score, FPG, HbA1C and FLI, only HbA1C added a significant prediction information (AUROC: 72.8% for full model vs 69.4% for the model without HbA1C; p = 0.028), while the removal of FLI to the full model did not alter its predictive value (AUROC 72.2%). The predictive value for NOD conversion was not significantly better for HOMA-IR compared to FLI (AUROC: 69.3 vs 63.7%, p = 0.067). CONCLUSIONS: FLI is a simple, practical score to further stratify the risk of conversion to NOD or the possibility of prediabetes reversion in clinical practice, independently of classical glucose parameters. TRIAL REGISTRATION: ClincialTrials.gov number NCT01218061 and NCT01432509.

Disturbance of apolipoprotein B100 containing lipoprotein metabolism in severe hyperlipidemic and lipodystrophic HIV patients on combined antiretroviral therapy: evidences of insulin resistance effect.

The aim was to study the mechanisms involved in the dyslipidemia associated with lipodystrophy in HIV infected patients on antiretroviral therapy (ART). We investigated the in vivo kinetics of apolipoprotein B100 (apoB) containing lipoproteins using a 14 h primed constant infusion of [5,5,5, (2)H(3)] leucine and compartmental modelling in normolipidemic without lipodystrophy (7 patients, NLD) or dyslipidemic with lipodystrophy (7 patients, LD) treated with ART. Subjects in group LD showed higher plasma triglycerides (5.73+/-3.58 vs 1.29+/-0.54 g/L, p<0.005), total cholesterol (2.98+/-0.95 vs 1.74+/-0.26 g/L, p<0.05), apoB (1.49+/-1.11 vs 0.51+/-0.11 g/L, p<0.005) and apolipoprotein CIII in apoB containing lipoproteins (117.7+/-42.2 vs 22.6+/-23.9 g/L, p<0.005). LD subjects exhibited an insulin resistant as observed by higher HOMA (3.44+/-1.62 vs 1.60+/-0.61, p<0.05). They exhibited an increase in VLDL (1.24+/-0.33 vs 0.80+/-0.21 mg/kg/h, p<0.05), decrease in IDL (0.20+/-0.10 vs 0.48+/-0.24 mg/kg/h, p<0.05) and no difference in LDL (0.38+/-0.19 vs 0.45+/-0.25 mg/kg/h) production rate. LD subject also showed a dramatic decrease in transformation of VLDL to IDL (0.013+/-0.010 vs 0.258+/-0.206 h(-1), p<0.005) and IDL to LDL (0.088+/-0.093 vs 0.366+/-0.189 h(-1), p<0.05) and a decrease in fractional catabolic rate (FCR) of VLDL (0.199+/-0.132 vs 0.555+/-0.398 h(-1), p<0.05), IDL (0.110+/-0.08 vs 0.523+/-0.275 h(-1), p<0.05) and LDL (0.010+/-0.005 vs 0.025+/-0.014 h(-1), p<0.05). These disturbances, overproduction and an overall delayed catabolism of apoB, are similar to those observed using the same protocol in insulin resistant subjects. Our study suggests that metabolic disturbance of apoB100 observed in lipodystrophic HIV in combined antiretroviral therapy are consecutive to insulin resistance induced by the treatment.

Apolipoprotein B100 metabolism in autosomal-dominant hypercholesterolemia related to mutations in PCSK9.

OBJECTIVE: We have reported further heterogeneity in familial autosomal-dominant hypercholesterolemia (FH) related to mutation in proprotein convertase subtilisin/kexin type 9 (PCSK9) gene previously named neural apoptosis regulated convertase 1 (Narc-1). Our aim was to define the metabolic bases of this new form of hypercholesterolemia. METHODS AND RESULTS: In vivo kinetics of apolipoprotein B100-containing lipoproteins using a 14-hour primed constant infusion of [2H3] leucine was conducted in 2 subjects carrying the mutation S127R in PCSK9, controls subjects, and FH subjects with known mutations on the low-density lipoprotein (LDL) receptor gene (LDL-R). Apo B100 production, catabolism, and transfer rates were estimated from very LDL (VLDL), intermediate-density lipoprotein (IDL), and LDL tracer enrichments by compartmental analysis. PCSK9 mutation dramatically increased the production rate of apolipoprotein B100 (3-fold) compared with controls or LDL-R mutated subjects, related to direct overproduction of VLDL (3-fold), IDL (3-fold), and LDL (5-fold). The 2 subjects also showed a decrease in VLDL and IDL conversion (10% to 30% of the controls). LDL fractional catabolic rate was slightly decreased (by 30%) compared with controls but still higher than LDL-R-mutated subjects. CONCLUSIONS: These results showed that the effect of the S127R mutation of PCSK9 on plasma cholesterol homeostasis is mainly related to an overproduction of apolipoprotein B100.

Laparoscopic Gastric Banding in Obese Patients with Sleep Apnea: A 3-Year Controlled Study and Follow-up After 10 Years.

BACKGROUND: The purpose of this study was to compare the effects of intensive nutritional care (INC) and laparoscopic adjustable gastric banding (LAGB) on nocturnal non-invasive ventilation (NIV) requirement in obese patients using short-, medium-, and long-term follow-up data. METHODS: This prospective randomized controlled trial included obese patients with obstructive sleep apnea (OSA) treated by NIV. Patients were randomized to the INC and LAGB groups. The primary endpoint was the theoretical rate of weaning from NIV at years 1 and 3. Data were also collected from patients 10 years after randomization. RESULTS: Sixty-three patients were randomized. The rate of weaning from NIV did not differ significantly between the LAGB and INC groups at year 1 (35 vs. 13%) or year 3 (14 vs. 21%). Percentages of excess weight loss were greater in the LAGB group than in the INC group at years 1 (33 vs. 15%, p = 0.002) and 3 (27 vs. 8%, p = 0.014). Decreases in the apnea-hypopnea index were observed in the LAGB group from baseline to year 1 (-44%, p = 0.001) and from baseline to year 3 (-26%, p = 0.044). After 10 years, the weaning rate was low and similar between groups. CONCLUSION: LAGB was not superior to INC for weaning from NIV at 1 and 3 years in obese patients with OSA.

Characterization of Autosomal Dominant Hypercholesterolemia Caused by PCSK9 Gain of Function Mutations and Its Specific Treatment With Alirocumab, a PCSK9 Monoclonal Antibody.

BACKGROUND: Patients with PCSK9 gene gain of function (GOF) mutations have a rare form of autosomal dominant hypercholesterolemia. However, data examining their clinical characteristics and geographic distribution are lacking. Furthermore, no randomized treatment study in this population has been reported. METHODS AND RESULTS: We compiled clinical characteristics of PCSK9 GOF mutation carriers in a multinational retrospective, cross-sectional, observational study. We then performed a randomized placebo-phase, double-blind study of alirocumab 150 mg administered subcutaneously every 2 weeks to 13 patients representing 4 different PCSK9 GOF mutations with low-density lipoprotein cholesterol (LDL-C) ≥70 mg/dL on their current lipid-lowering therapies at baseline. Observational study: among 164 patients, 16 different PCSK9 GOF mutations distributed throughout the gene were associated with varying severity of untreated LDL-C levels. Coronary artery disease was common (33%; average age of onset, 49.4 years), and untreated LDL-C concentrations were higher compared with matched carriers of mutations in the LDLR (n=2126) or apolipoprotein B (n=470) genes. Intervention study: in PCSK9 GOF mutation patients randomly assigned to receive alirocumab, mean percent reduction in LDL-C at 2 weeks was 62.5% (P<0.0001) from baseline, 53.7% compared with placebo-treated PCSK9 GOF mutation patients (P=0.0009; primary end point). After all subjects received 8 weeks of alirocumab treatment, LDL-C was reduced by 73% from baseline (P<0.0001). CONCLUSIONS: PCSK9 GOF mutation carriers have elevated LDL-C levels and are at high risk of premature cardiovascular disease. Alirocumab, a PCSK9 antibody, markedly lowers LDL-C levels and seems to be well tolerated in these patients. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique Identifier: NCT01604824.

Dual peroxisome proliferator-activated receptor α/δ agonist GFT505 improves hepatic and peripheral insulin sensitivity in abdominally obese subjects.

OBJECTIVE: The development of new insulin sensitizers is an unmet need for the treatment of type 2 diabetes. We investigated the effect of GFT505, a dual peroxisome proliferator-activated receptor (PPAR)-α/δ agonist, on peripheral and hepatic insulin sensitivity. RESEARCH DESIGN AND METHODS: Twenty-two abdominally obese insulin-resistant males (homeostasis model assessment of insulin resistance>3) were randomly assigned in a randomized crossover study to subsequent 8-week treatment periods with GFT505 (80 mg/day) or placebo, followed by a two-step hyperinsulinemic-euglycemic insulin clamp with a glucose tracer to calculate endogenous glucose production (EGP). The primary end point was the improvement in glucose infusion rate (GIR). Gene expression analysis was performed on skeletal muscle biopsy specimens. RESULTS: GFT505 improved peripheral insulin sensitivity, with a 21% (P=0.048) increase of the GIR at the second insulin infusion period. GFT505 also enhanced hepatic insulin sensitivity, with a 44% (P=0.006) increase of insulin suppression of EGP at the first insulin infusion period. Insulin-suppressed plasma free fatty acid concentrations were significantly reduced on GFT505 treatment (0.21±0.07 vs. 0.27±0.11 mmol/L; P=0.006). Neither PPARα nor PPARδ target genes were induced in skeletal muscle, suggesting a liver-targeted action of GFT505. GFT505 significantly reduced fasting plasma triglycerides (-21%; P=0.003) and LDL cholesterol (-13%; P=0.0006), as well as liver enzyme concentrations (γ-glutamyltranspeptidase: -30.4%, P=0.003; alanine aminotransferase: -20.5%, P=0.004). There was no safety concern or any indication of PPARγ activation with GFT505. CONCLUSIONS: The dual PPARα/δ agonist GFT505 is a liver-targeted insulin-sensitizer that is a promising drug candidate for the treatment of type 2 diabetes and nonalcoholic fatty liver disease.

Plasma PCSK9 concentrations during an oral fat load and after short term high-fat, high-fat high-protein and high-fructose diets.

BACKGROUND: 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. METHODS: 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). FINDINGS: 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. CONCLUSIONS: 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.

Effects of the new dual PPAR α/δ agonist GFT505 on lipid and glucose homeostasis in abdominally obese patients with combined dyslipidemia or impaired glucose metabolism.

OBJECTIVE: We evaluated the metabolic effects and tolerability of GFT505, a novel dual peroxisome proliferator-activated receptor α/δ agonist, in abdominally obese patients with either combined dyslipidemia or prediabetes. RESEARCH DESIGN AND METHODS: The S1 study was conducted in 94 patients with combined dyslipidemia while the S2 study was conducted in 47 patients with prediabetes. Participants were randomly assigned in a double-blind manner to GFT505 at 80 mg/day or placebo for 28 (S1) or 35 (S2) days. Primary efficacy end points were changes from baseline at week 4 in both fasting plasma triglycerides and HDL cholesterol in the S1 group and 2-h glucose upon oral glucose tolerance test in the S2 group. RESULTS: In comparison with placebo, GFT505 significantly reduced fasting plasma triglycerides (S1: least squares means -16.7% [95% one-sided CI -∞ to -5.3], P = 0.005; S2: -24.8% [-∞ to -10.5], P = 0.0003) and increased HDL cholesterol (S1: 7.8% [3.0 to ∞], P = 0.004; S2: 9.3% [1.7 to ∞], P = 0.009) in both studies, whereas LDL cholesterol only decreased in S2 (-11.0% [ -∞ to -3.5], P = 0.002). In S2, GFT505 did not reduce 2-h glucose (-0.52 mmol/L [-∞ to 0.61], P = 0.18) but led to a significant decrease of homeostasis model assessment of insulin resistance (-31.4% [-∞ to 12.5], P = 0.001), fasting plasma glucose (-0.37 mmol/L [-∞ to -0.10], P = 0.01) and fructosamine (-3.6% [-∞ to -0.20], P = 0.02). GFT505 also reduced γ glutamyl transferase levels in both studies (S1: -19.9% [-∞ to -12.8], P < 0.0001; S2: -15.1% [-∞ to -1.1], P = 0.004). No specific adverse safety signals were reported during the studies. CONCLUSIONS: GFT505 may be considered a new drug candidate for the treatment of lipid and glucose disorders associated with the metabolic syndrome.

Fasting plasma chenodeoxycholic acid and cholic acid concentrations are inversely correlated with insulin sensitivity in adults.

BACKGROUND: Accumulating data suggest a novel role for bile acids (BAs) in modulating metabolic homeostasis. BA treatment has been shown to improve glucose tolerance and to increase energy expenditure in mice. Here, we investigated the relationship between fasting plasma BAs concentrations and metabolic parameters in humans. FINDINGS: Fasting plasma glucose, insulin and lipid profile were measured in 14 healthy volunteers, 20 patients with type 2 diabetes (T2D), and 22 non-diabetic abdominally obese subjects. Insulin sensitivity was also assessed by the determination of the glucose infusion rate (GIR) during a hyperinsulinemic-euglycemic clamp in a subgroup of patients (9 healthy and 16 T2D subjects). Energy expenditure was measured by indirect calorimetry. Plasma cholic acid (CA), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) concentrations were analyzed by gas chromatograph-mass spectrometry. In univariable analysis, a positive association was found between HOMA-IR and plasma CDCA (ß = 0.09, p = 0.001), CA (ß = 0.03, p = 0.09) and DCA concentrations (ß = 0.07, p < 0.0001). Spearman analysis retrieved an inverse relationship between plasma CDCA (r = -0.44, p = 0.03), CA (r = -0.65, p = 0.001) and the GIR. HOMA-IR remained positively associated with CDCA (ß = 0.11, p = 0.01), CA (ß = 0.04, p = 0.01) and DCA (ß = 0.06, p = 0.007) in multivariable analysis, after adjustment for age, gender, BMI, HbA1C and plasma lipid parameters. In contrast, HbA1c, energy expenditure and plasma lipid concentrations were not correlated with plasma BAs levels in multivariable analysis. CONCLUSIONS: Both plasma CDCA, CA and DCA concentrations were negatively associated with insulin sensitivity in a wide range of subjects.

Acetogenic fibers reduce fasting glucose turnover but not peripheral insulin resistance in metabolic syndrome patients.

BACKGROUND & AIMS: The acute ingestion of an acetogenic indigestible carbohydrate (lactulose) increased acetate turnover associated with decreased lipolysis (glycerol turnover) in insulin-resistant patients. It is not known whether a decreased lipolysis by chronic ingestion of acetogenic indigestible carbohydrates or fibers improves glucose turnover and insulin sensitivity. METHODS: Twenty-one men with metabolic syndrome ingested daily standardized drinks, with or without 28 g acetogenic fibers (acacia gum and pectin), for 5 weeks in a randomized double-blind crossover controlled study design. Euglycaemic-hyperinsulinaemic (EH) clamps coupled with kinetic studies were performed in the fasting state after treatments. RESULTS: Flatulence was more frequent with fiber treatment. Body weight, lipids as well as acetate and glycerol turnovers were unchanged. Fasting endogenous glucose turnover was improved after fiber treatment (7.9 ± 1.3 µmol kg(-1) min(-1)) compared with control (8.6 ± 1.6 µmol kg(-1) min(-1), P < 0.05). But insulin sensitivity (glucose infusion rate) during the EH clamp was not different at the end of fiber and control treatments, 3.7 ± 1.8 and 3.8 ± 1.5 mg kg(-1) min(-1), respectively, nor fasting plasma glucose and insulin. CONCLUSIONS: The chronic ingestion of acacia gum and pectin fibers did not decrease lipolysis but improved fasting endogenous glucose turnover with no effect on peripheral insulin resistance in metabolic syndrome patients.

A fourth locus for autosomal dominant hypercholesterolemia maps at 16q22.1.

Autosomal dominant hypercholesterolemia (ADH) is characterized by isolated increase in plasmatic low-density lipoprotein (LDL) cholesterol levels associated with high risk of premature cardiovascular disease. Mutations in LDLR, APOB, and PCSK9 genes have been shown to cause ADH. We now report further genetic heterogeneity of ADH through the study of a large French family in which the involvement of these three genes was excluded. A genome-wide scan mapped the disease-causing gene, named HCHOLA4, at 16q22.1 in a 7.89-Mb interval containing 154 genes with a maximum LOD score of 3.9. To reduce the linked region, we genotyped 18 smaller non-LDLR/non-APOB/non-PCSK9-ADH families at the HCHOLA4 locus. Six families did not exclude linkage to the locus, but none allowed reduction of the disease interval. The 154 regional genes were sorted according to the function of the encoded protein and tissue expression profiles, and 57 genes were analyzed through sequencing of their coding region and close flanking intronic parts. No disease-causing mutation was identified in these families, particularly in the LCAT gene. Finally, our results also show the existence of other ADH genes as nine families were neither linked to LDLR, APOB, and PCSK9 genes nor to the new HCHOLA4 locus.