ANGPTL3 Inhibition With Evinacumab Results in Faster Clearance of IDL and LDL apoB in Patients With Homozygous Familial Hypercholesterolemia-Brief Report.

[Figure: see text].

Taking One Step Back in Familial Hypercholesterolemia: STAP1 Does Not Alter Plasma LDL (Low-Density Lipoprotein) Cholesterol in Mice and Humans.

OBJECTIVE: STAP1, encoding for STAP1 (signal transducing adaptor family member 1), has been reported as a candidate gene associated with familial hypercholesterolemia. Unlike established familial hypercholesterolemia genes, expression of STAP1 is absent in liver but mainly observed in immune cells. In this study, we set out to validate STAP1 as a familial hypercholesterolemia gene. Approach and Results: A whole-body Stap1 knockout mouse model (Stap1-/-) was generated and characterized, without showing changes in plasma lipid levels compared with controls. In follow-up studies, bone marrow from Stap1-/- mice was transplanted to Ldlr-/- mice, which did not show significant changes in plasma lipid levels or atherosclerotic lesions. To functionally assess whether STAP1 expression in B cells can affect hepatic function, HepG2 cells were cocultured with peripheral blood mononuclear cells isolated from heterozygotes carriers of STAP1 variants and controls. The peripheral blood mononuclear cells from STAP1 variant carriers and controls showed similar LDLR mRNA and protein levels. Also, LDL (low-density lipoprotein) uptake by HepG2 cells did not differ upon coculturing with peripheral blood mononuclear cells isolated from either STAP1 variant carriers or controls. In addition, plasma lipid profiles of 39 carriers and 71 family controls showed no differences in plasma LDL cholesterol, HDL (high-density lipoprotein) cholesterol, triglycerides, and lipoprotein(a) levels. Similarly, B-cell populations did not differ in a group of 10 STAP1 variant carriers and 10 age- and sex-matched controls. Furthermore, recent data from the UK Biobank do not show association between STAP1 rare gene variants and LDL cholesterol. CONCLUSIONS: Our combined studies in mouse models and carriers of STAP1 variants indicate that STAP1 is not a familial hypercholesterolemia gene.

Oral butyrate does not affect innate immunity and islet autoimmunity in individuals with longstanding type 1 diabetes: a randomised controlled trial.

AIMS/HYPOTHESIS: The pathophysiology of type 1 diabetes has been linked to altered gut microbiota and more specifically to a shortage of intestinal production of the short-chain fatty acid (SCFA) butyrate, which may play key roles in maintaining intestinal epithelial integrity and in human and gut microbial metabolism. Butyrate supplementation can protect against autoimmune diabetes in mouse models. We thus set out to study the effect of oral butyrate vs placebo on glucose regulation and immune variables in human participants with longstanding type 1 diabetes. METHODS: We administered a daily oral dose of 4 g sodium butyrate or placebo for 1 month to 30 individuals with longstanding type 1 diabetes, without comorbidity or medication use, in a randomised (1:1), controlled, double-blind crossover trial, with a washout period of 1 month in between. Participants were randomly allocated to the 'oral sodium butyrate capsules first' or 'oral placebo capsules first' study arm in blocks of five. The clinical investigator received blinded medication from the clinical trial pharmacy. All participants, people doing measurements or examinations, or people assessing the outcomes were blinded to group assignment. The primary outcome was a change in the innate immune phenotype (monocyte subsets and in vitro cytokine production). Secondary outcomes were changes in blood markers of islet autoimmunity (cell counts, lymphocyte stimulation indices and CD8 quantum dot assays), glucose and lipid metabolism, beta cell function (by mixed-meal test), gut microbiota and faecal SCFA. The data was collected at the Amsterdam University Medical Centers. RESULTS: All 30 participants were analysed. Faecal butyrate and propionate levels were significantly affected by oral butyrate supplementation and butyrate treatment was safe. However, this modulation of intestinal SCFAs did not result in any significant changes in adaptive or innate immunity, or in any of the other outcome variables. In our discussion, we elaborate on this important discrepancy with previous animal work. CONCLUSIONS/INTERPRETATION: Oral butyrate supplementation does not significantly affect innate or adaptive immunity in humans with longstanding type 1 diabetes. TRIAL REGISTRATION: Netherlands Trial Register: NL4832 (www.trialregister.nl). DATA AVAILABILITY: Raw sequencing data are available in the European Nucleotide Archive repository (https://www.ebi.ac.uk/ena/browse) under study PRJEB30292. FUNDING: The study was funded by a Le Ducq consortium grant, a CVON grant, a personal ZONMW-VIDI grant and a Dutch Heart Foundation grant.

N-Glycosylation Defects in Humans Lower Low-Density Lipoprotein Cholesterol Through Increased Low-Density Lipoprotein Receptor Expression.

BACKGROUND: The importance of protein glycosylation in regulating lipid metabolism is becoming increasingly apparent. We set out to further investigate this by studying patients with type I congenital disorders of glycosylation (CDGs) with defective N-glycosylation. METHODS: We studied 29 patients with the 2 most prevalent types of type I CDG, ALG6 (asparagine-linked glycosylation protein 6)-deficiency CDG and PMM2 (phosphomannomutase 2)-deficiency CDG, and 23 first- and second-degree relatives with a heterozygous mutation and measured plasma cholesterol levels. Low-density lipoprotein (LDL) metabolism was studied in 3 cell models-gene silencing in HepG2 cells, patient fibroblasts, and patient hepatocyte-like cells derived from induced pluripotent stem cells-by measuring apolipoprotein B production and secretion, LDL receptor expression and membrane abundance, and LDL particle uptake. Furthermore, SREBP2 (sterol regulatory element-binding protein 2) protein expression and activation and endoplasmic reticulum stress markers were studied. RESULTS: We report hypobetalipoproteinemia (LDL cholesterol [LDL-C] and apolipoprotein B below the fifth percentile) in a large cohort of patients with type I CDG (mean age, 9 years), together with reduced LDL-C and apolipoprotein B in clinically unaffected heterozygous relatives (mean age, 46 years), compared with 2 separate sets of age- and sex-matched control subjects. ALG6 and PMM2 deficiency led to markedly increased LDL uptake as a result of increased cell surface LDL receptor abundance. Mechanistically, this outcome was driven by increased SREBP2 protein expression accompanied by amplified target gene expression, resulting in higher LDL receptor protein levels. Endoplasmic reticulum stress was not found to be a major mediator. CONCLUSIONS: Our study establishes N-glycosylation as an important regulator of LDL metabolism. Given that LDL-C was also reduced in a group of clinically unaffected heterozygotes, we propose that increasing LDL receptor-mediated cholesterol clearance by targeting N-glycosylation in the LDL pathway may represent a novel therapeutic strategy to reduce LDL-C and cardiovascular disease.

The effect of dapagliflozin on apolipoprotein B and glucose fluxes in patients with type 2 diabetes and well-controlled plasma LDL cholesterol.

AIM: To dissect the effects of the sodium-glucose linked transporter 2 inhibitor dapagliflozin on lipid metabolism and assess whether these effects could potentially offset cardiovascular benefit with this drug-class. MATERIALS AND METHODS: We assessed the effect of dapagliflozin on lipid metabolism in 11 adults with uncomplicated type 2 diabetes. After 4 weeks of statin wash-out and 4 weeks of rosuvastatin 10 mg treatment, participants were treated with dapagliflozin 10 mg once-daily for 5 weeks. Before and after dapagliflozin, plasma lipids were measured and very low-density lipoprotein (VLDL)-1 and VLDL-2 apolipoprotein (Apo)B fluxes were assessed using (5.5.5-2 H3 )-leucine tracer infusion. In addition, hepatic and peripheral insulin sensitivity as well as insulin-mediated inhibition of peripheral lipolysis were measured during a two-step hyperinsulinemic-euglycaemic clamp using (6,6-2 H2 )-glucose and (1,1,2,3,3-2 H5 )-glycerol tracers. RESULTS: Rosuvastatin decreased all plasma lipids significantly: total cholesterol from 4.5 (3.2-6.2) to 3.1 (2.5-3.8) mmol/L, LDL cholesterol from 2.6 (1.7-3.4) to 1.5 (1.1-2.2) mmol/L, HDL cholesterol from 1.34 (0.80-2.02) to 1.19 (0.74-1.89) mmol/L and triglycerides from 0.92 (0.31-3.91) to 0.79 (0.32-2.10) mmol/L. The addition of dapaglifozin to rosuvastatin did not raise either LDL cholesterol or total cholesterol, and only increased HDL cholesterol by 0.08 (-0.03-0.13) mmol/L (P = 0.03). In line with this, dapagliflozin did not affect VLDL-1 or VLDL-2 ApoB fluxes. Fasting endogenous glucose production tended to increase by 0.9 (-3.4-3.1) µmol kg-1 min-1 (P = 0.06), but no effect on hepatic and peripheral insulin sensitivity or on peripheral lipolysis was observed. CONCLUSIONS: Dapagliflozin has no effect on plasma LDL-cholesterol levels or VLDL-apoB fluxes in the context of optimal lipid-lowering treatment, which will thus not limit cardiovascular benefit when lipids are adequately controlled.

Reduced CETP glycosylation and activity in patients with homozygous B4GALT1 mutations.

The importance of protein glycosylation in regulating lipid metabolism is becoming increasingly apparent. We set out to further investigate this by studying the effects of defective glycosylation on plasma lipids in patients with B4GALT1-CDG, caused by a mutation in B4GALT1 with defective N-linked glycosylation. We studied plasma lipids, cholesteryl ester transfer protein (CETP) glyco-isoforms with isoelectric focusing followed by a western blot and CETP activity in three known B4GALT1-CDG patients and compared them with 11 age- and gender-matched, healthy controls. B4GALT1-CDG patients have significantly lowered non-high density lipoprotein cholesterol (HDL-c) and total cholesterol to HDL-c ratio compared with controls and larger HDL particles. Plasma CETP was hypoglycosylated and less active in B4GALT1-CDG patients compared to matched controls. Our study provides insight into the role of protein glycosylation in human lipoprotein homeostasis. The hypogalactosylated, hypo-active CETP found in patients with B4GALT1-CDG indicates a role of protein galactosylation in regulating plasma HDL and LDL. Patients with B4GALT1-CDG have large HDL particles probably due to hypogalactosylated, hypo-active CETP.

Naturally Occurring Variants in LRP1 (Low-Density Lipoprotein Receptor-Related Protein 1) Affect HDL (High-Density Lipoprotein) Metabolism Through ABCA1 (ATP-Binding Cassette A1) and SR-B1 (Scavenger Receptor Class B Type 1) in Humans.

OBJECTIVE: Studies into the role of LRP1 (low-density lipoprotein receptor-related protein 1) in human lipid metabolism are scarce. Although it is known that a common variant in LRP1 (rs116133520) is significantly associated with HDL-C (high-density lipoprotein cholesterol), the mechanism underlying this observation is unclear. In this study, we set out to study the functional effects of 2 rare LRP1 variants identified in subjects with extremely low HDL-C levels. APPROACH AND RESULTS: In 2 subjects with HDL-C below the first percentile for age and sex and moderately elevated triglycerides, we identified 2 rare variants in LRP1: p.Val3244Ile and p.Glu3983Asp. Both variants decrease LRP1 expression and stability. We show in a series of translational experiments that these variants culminate in reduced trafficking of ABCA1 (ATP-binding cassette A1) to the cell membrane. This is accompanied by an increase in cell surface expression of SR-B1 (scavenger receptor class B type 1). Combined these effects may contribute to low HDL-C levels in our study subjects. Supporting these findings, we provide epidemiological evidence that rs116133520 is associated with apo (apolipoprotein) A1 but not with apoB levels. CONCLUSIONS: This study provides the first evidence that rare variants in LRP1 are associated with changes in human lipid metabolism. Specifically, this study shows that LRP1 may affect HDL metabolism by virtue of its effect on both ABCA1 and SR-B1.

ABCA8 Regulates Cholesterol Efflux and High-Density Lipoprotein Cholesterol Levels.

OBJECTIVE: High-density lipoproteins (HDL) are considered to protect against atherosclerosis in part by facilitating the removal of cholesterol from peripheral tissues. However, factors regulating lipid efflux are incompletely understood. We previously identified a variant in adenosine triphosphate-binding cassette transporter A8 (ABCA8) in an individual with low HDL cholesterol (HDLc). Here, we investigate the role of ABCA8 in cholesterol efflux and in regulating HDLc levels. APPROACH AND RESULTS: We sequenced ABCA8 in individuals with low and high HDLc and identified, exclusively in low HDLc probands, 3 predicted deleterious heterozygous ABCA8 mutations (p.Pro609Arg [P609R], IVS17-2 A>G and p.Thr741Stop [T741X]). HDLc levels were lower in heterozygous mutation carriers compared with first-degree family controls (0.86±0.34 versus 1.17±0.26 mmol/L; P=0.005). HDLc levels were significantly decreased by 29% (P=0.01) in Abca8b-/- mice on a high-cholesterol diet compared with wild-type mice, whereas hepatic overexpression of human ABCA8 in mice resulted in significant increases in plasma HDLc and the first steps of macrophage-to-feces reverse cholesterol transport. Overexpression of wild-type but not mutant ABCA8 resulted in a significant increase (1.8-fold; P=0.01) of cholesterol efflux to apolipoprotein AI in vitro. ABCA8 colocalizes and interacts with adenosine triphosphate-binding cassette transporter A1 and further potentiates adenosine triphosphate-binding cassette transporter A1-mediated cholesterol efflux. CONCLUSIONS: ABCA8 facilitates cholesterol efflux and modulates HDLc levels in humans and mice.

Nile Red Quantifier: a novel and quantitative tool to study lipid accumulation in patient-derived circulating monocytes using confocal microscopy.

The inflammatory profile of circulating monocytes is an important biomarker for atherosclerotic plaque vulnerability. Recent research revealed that peripheral lipid uptake by monocytes alters their phenotype toward an inflammatory state and this coincides with an increased lipid droplet (LD) content. Determination of lipid content of circulating monocytes is, however, not very well established. Based on Nile Red (NR) neutral LD imaging, using confocal microscopy and computational analysis, we developed NR Quantifier (NRQ), a novel quantification method to assess LD content in monocytes. Circulating monocytes were isolated from blood and used for the NR staining procedure. In monocytes stained with NR, we clearly distinguished, based on 3D imaging, phospholipids and exclusively intracellular neutral lipids. Next, we developed and validated NRQ, a semi-automated quantification program that detects alterations in lipid accumulation. NRQ was able to detect LD alterations after ex vivo exposure of isolated monocytes to freshly isolated LDL in a time- and dose-dependent fashion. Finally, we validated NRQ in patients with familial hypercholesterolemia and obese subjects in pre- and postprandial state. In conclusion, NRQ is a suitable tool to detect even small differences in neutral LD content in circulating monocytes using NR staining.

Role of sulfatase 2 in lipoprotein metabolism and angiogenesis.

PURPOSE OF REVIEW: This article summarizes the current evidence to support a role of sulfatase 2 (SULF2) in triglyceride-rich lipoprotein (TRL) metabolism and angiogenesis. RECENT FINDINGS: Heparan sulfate proteoglycans (HSPG) are involved in the hepatic clearance of TRLs in mice and in humans. Different genetically modified mouse models have been instrumental to provide evidence that syndecan1, the core protein of HSPG, but also the degree of sulfation of the heparin sulfate chain, attached to syndecan 1, is important for hepatic TRL metabolism. Studies in humans demonstrate the regulating role of SULF2 in the hepatic uptake of TRL by HSPG and demonstrate the importance of 6-O-sulfation, modulated by SULF2, for HSPG function. The role of SULF2 in angiogenesis is illustrated by increased SULF2 mRNA expression in the stalk cells of angiogenic vascular sprouts that use fatty acids derived from TRL as a source for biomass production. Interestingly, SULF2 also interferes with HSPG-vascular endothelial growth factor binding, which impacts upon the angiogenic properties of stalk cells. SUMMARY: SULF2 is a multifaceted protein involved in TRL homeostasis and angiogenesis. Future investigations should focus on the potential benefits of targeting SULF2 in atherosclerosis and angiogenesis.

Mutations in STAP1 are associated with autosomal dominant hypercholesterolemia.

RATIONALE: Autosomal-dominant hypercholesterolemia (ADH) is characterized by elevated low-density lipoprotein cholesterol levels and increased risk for coronary vascular disease. ADH is caused by mutations in the low-density lipoprotein receptor, apolipoprotein B, or proprotein convertase subtilisin/kexin 9. A number of patients, however, suffer from familial hypercholesterolemia 4 (FH4), defined as ADH in absence of mutations in these genes and thereafter use the abbreviation FH4. OBJECTIVE: To identify a fourth locus associated with ADH. METHODS AND RESULTS: Parametric linkage analysis combined with exome sequencing in a FH4 family resulted in the identification of the variant p.Glu97Asp in signal transducing adaptor family member 1 (STAP1), encoding signal transducing adaptor family member 1. Sanger sequencing of STAP1 in 400 additional unrelated FH4 probands identified a second p.Glu97Asp carrier and 3 additional missense variants, p.Leu69Ser, p.Ile71Thr, and p.Asp207Asn. STAP1 carriers (n=40) showed significantly higher plasma total cholesterol and low-density lipoprotein cholesterol levels compared with nonaffected relatives (n=91). CONCLUSIONS: We mapped a novel ADH locus at 4p13 and identified 4 variants in STAP1 that associate with ADH.

Liposomal prednisolone promotes macrophage lipotoxicity in experimental atherosclerosis.

Atherosclerosis is a lipid-driven inflammatory disease, for which nanomedicinal interventions are under evaluation. Previously, we showed that liposomal nanoparticles loaded with prednisolone (LN-PLP) accumulated in plaque macrophages, however, induced proatherogenic effects in patients. Here, we confirmed in low-density lipoprotein receptor knockout (LDLr(-/-)) mice that LN-PLP accumulates in plaque macrophages. Next, we found that LN-PLP infusions at 10mg/kg for 2weeks enhanced monocyte recruitment to plaques. In follow up, after 6weeks of LN-PLP exposure we observed (i) increased macrophage content, (ii) more advanced plaque stages, and (iii) larger necrotic core sizes. Finally, in vitro studies showed that macrophages become lipotoxic after LN-PLP exposure, exemplified by enhanced lipid loading, ER stress and apoptosis. These findings indicate that liposomal prednisolone may paradoxically accelerate atherosclerosis by promoting macrophage lipotoxicity. Hence, future (nanomedicinal) drug development studies are challenged by the multifactorial nature of atherosclerotic inflammation.

Triglyceride-Rich Lipoproteins and Remnants: Targets for Therapy?

It is now evident that elevated circulating levels of triglycerides in the non-fasting state, a marker for triglyceride (TG)-rich remnant particles, are associated with increased risk of premature cardiovascular disease (CVD). Recent findings from basic and clinical studies have begun to elucidate the mechanisms that contribute to the atherogenicity of these apoB-containing particles. Here, we review current knowledge of the formation, intravascular remodelling and catabolism of TG-rich lipoproteins and highlight (i) the pivotal players involved in this process, including lipoprotein lipase, glycosylphosphatidylinositol HDL binding protein 1 (GPIHBP1), apolipoprotein (apo) C-II, apoC-III, angiopoietin-like protein (ANGPTL) 3, 4 and 8, apoA-V and cholesteryl ester transfer protein; (ii) key determinants of triglyceride (TG) levels and notably rates of production of very-low-density lipoprotein 1 (VLDL1) particles; and (iii) the mechanisms which underlie the atherogenicity of remnant particles. Finally, we emphasise the polygenic nature of moderate hypertriglyceridemia and briefly discuss modalities for its clinical management. Several new therapeutic strategies to attenuate hypertriglyceridemia have appeared recently, among which those targeted to apoC-III appear to hold considerable promise.

The endoplasmic reticulum coat protein II transport machinery coordinates cellular lipid secretion and cholesterol biosynthesis.

Triglycerides and cholesterol are essential for life in most organisms. Triglycerides serve as the principal energy storage depot and, where vascular systems exist, as a means of energy transport. Cholesterol is essential for the functional integrity of all cellular membrane systems. The endoplasmic reticulum is the site of secretory lipoprotein production and de novo cholesterol synthesis, yet little is known about how these activities are coordinated with each other or with the activity of the COPII machinery, which transports endoplasmic reticulum cargo to the Golgi. The Sar1B component of this machinery is mutated in chylomicron retention disorder, indicating that this Sar1 isoform secures delivery of dietary lipids into the circulation. However, it is not known why some patients with chylomicron retention disorder develop hepatic steatosis, despite impaired intestinal fat malabsorption, and why very severe hypocholesterolemia develops in this condition. Here, we show that Sar1B also promotes hepatic apolipoprotein (apo) B lipoprotein secretion and that this promoting activity is coordinated with the processes regulating apoB expression and the transfer of triglycerides/cholesterol moieties onto this large lipid transport protein. We also show that although Sar1A antagonizes the lipoprotein secretion-promoting activity of Sar1B, both isoforms modulate the expression of genes encoding cholesterol biosynthetic enzymes and the synthesis of cholesterol de novo. These results not only establish that Sar1B promotes the secretion of hepatic lipids but also adds regulation of cholesterol synthesis to Sar1B's repertoire of transport functions.

Increased large VLDL particles confer elevated cholesteryl ester transfer in diabetes.

BACKGROUND: Plasma cholesteryl ester transfer (CET), reflecting transfer of cholesteryl esters from high density lipoproteins (HDL) towards apolipoprotein B-containing lipoproteins, may promote atherosclerosis development, and is elevated in Type 2 diabetes mellitus (T2DM). We determined the extent to which the relationship of plasma CET with very low density lipoprotein (VLDL) and low density lipoprotein (LDL) subfractions is modified in T2DM. MATERIALS AND METHODS: Plasma CET, cholesteryl ester transfer protein (CETP) mass, as well as VLDL and LDL subfractions (nuclear magnetic resonance spectroscopy) were determined in 62 patients with T2DM and 53 nondiabetic subjects. RESULTS: Plasma CET and CETP mass were increased in T2DM, coinciding higher triglycerides and large VLDL particles (all P < 0·02). Plasma CET was positively related to the VLDL and the LDL particle concentration in age-, sex- and diabetes status-adjusted analysis (both P < 0·001). Multivariable linear regression analysis demonstrated an independent positive interaction between the presence of T2DM and the VLDL concentration on plasma CET (ß = 0·238, P = 0·033). The relationship of plasma CET with the VLDL concentration was also positively modified by plasma glucose (ß = 0·211, P = 0·004) and glycated haemoglobin (ß = 0·190, P = 0·012). Of the individual VLDL subfractions, a positive interaction of diabetes status with large VLDL on plasma CET was observed (ß = 0·280, P = 0·003). Neither the relationship of the LDL particle concentration nor of CETP mass with plasma CET was modified by the presence of T2DM (P > 0·15). CONCLUSION: Abnormalities in the concentration and composition of large VLDL particles are likely to contribute to elevated plasma CET in T2DM.

Prednisolone-containing liposomes accumulate in human atherosclerotic macrophages upon intravenous administration.

Drug delivery to atherosclerotic plaques via liposomal nanoparticles may improve therapeutic agents' risk-benefit ratios. Our paper details the first clinical studies of a liposomal nanoparticle encapsulating prednisolone (LN-PLP) in atherosclerosis. First, PLP's liposomal encapsulation improved its pharmacokinetic profile in humans (n=13) as attested by an increased plasma half-life of 63h (LN-PLP 1.5mg/kg). Second, intravenously infused LN-PLP appeared in 75% of the macrophages isolated from iliofemoral plaques of patients (n=14) referred for vascular surgery in a randomized, placebo-controlled trial. LN-PLP treatment did however not reduce arterial wall permeability or inflammation in patients with atherosclerotic disease (n=30), as assessed by multimodal imaging in a subsequent randomized, placebo-controlled study. In conclusion, we successfully delivered a long-circulating nanoparticle to atherosclerotic plaque macrophages in patients, whereas prednisolone accumulation in atherosclerotic lesions had no anti-inflammatory effect. Nonetheless, the present study provides guidance for development and imaging-assisted evaluation of future nanomedicine in atherosclerosis. FROM THE CLINICAL EDITOR: In this study, the authors undertook the first clinical trial using long-circulating liposomal nanoparticle encapsulating prednisolone in patients with atherosclerosis, based on previous animal studies. Despite little evidence of anti-inflammatory effect, the results have provided a starting point for future development of nanomedicine in cardiovascular diseases.

Impact of oral vancomycin on gut microbiota, bile acid metabolism, and insulin sensitivity.

BACKGROUND & AIMS: Obesity has been associated with changes in the composition and function of the intestinal microbiota. Modulation of the microbiota by antibiotics also alters bile acid and glucose metabolism in mice. Hence, we hypothesized that short term administration of oral antibiotics in humans would affect fecal microbiota composition and subsequently bile acid and glucose metabolism. METHODS: In this single blinded randomized controlled trial, 20 male obese subjects with metabolic syndrome were randomized to 7 days of amoxicillin 500 mg t.i.d. or 7 days of vancomycin 500 mg t.i.d. At baseline and after 1 week of therapy, fecal microbiota composition (Human Intestinal Tract Chip phylogenetic microarray), fecal and plasma bile acid concentrations as well as insulin sensitivity (hyperinsulinemic euglycemic clamp using [6,6-(2)H2]-glucose tracer) were measured. RESULTS: Vancomycin reduced fecal microbial diversity with a decrease of gram-positive bacteria (mainly Firmicutes) and a compensatory increase in gram-negative bacteria (mainly Proteobacteria). Concomitantly, vancomycin decreased fecal secondary bile acids with a simultaneous postprandial increase in primary bile acids in plasma (p<0.05). Moreover, changes in fecal bile acid concentrations were predominantly associated with altered Firmicutes. Finally, administration of vancomycin decreased peripheral insulin sensitivity (p<0.05). Amoxicillin did not affect any of these parameters. CONCLUSIONS: Oral administration of vancomycin significantly impacts host physiology by decreasing intestinal microbiota diversity, bile acid dehydroxylation and peripheral insulin sensitivity in subjects with metabolic syndrome. These data show that intestinal microbiota, particularly of the Firmicutes phylum contributes to bile acid and glucose metabolism in humans. This trial is registered at the Dutch Trial Register (NTR2566).

Genetic variant of the scavenger receptor BI in humans.

BACKGROUND: In mice, the scavenger receptor class B type I (SR-BI) is essential for the delivery of high-density lipoprotein (HDL) cholesterol to the liver and steroidogenic organs. Paradoxically, elevated HDL cholesterol levels are associated with increased atherosclerosis in SR-BI-knockout mice. It is unclear what role SR-BI plays in human metabolism. METHODS: We sequenced the gene encoding SR-BI in persons with elevated HDL cholesterol levels and identified a family with a new missense mutation (P297S). The functional effects of the P297S mutation on HDL binding, cellular cholesterol uptake and efflux, atherosclerosis, platelet function, and adrenal function were studied. RESULTS: Cholesterol uptake from HDL by primary murine hepatocytes that expressed mutant SR-BI was reduced to half of that of hepatocytes expressing wild-type SR-BI. Carriers of the P297S mutation had increased HDL cholesterol levels (70.4 mg per deciliter [1.8 mmol per liter], vs. 53.4 mg per deciliter [1.4 mmol per liter] in noncarriers; P<0.001) and a reduced capacity for efflux of cholesterol from macrophages, but the carotid artery intima-media thickness was similar in carriers and in family noncarriers. Platelets from carriers had increased unesterified cholesterol content and impaired function. In carriers, adrenal steroidogenesis was attenuated, as evidenced by decreased urinary excretion of sterol metabolites, a decreased response to corticotropin stimulation, and symptoms of diminished adrenal function. CONCLUSIONS: We identified a family with a functional mutation in SR-BI. The mutation carriers had increased HDL cholesterol levels and a reduction in cholesterol efflux from macrophages but no significant increase in atherosclerosis. Reduced SR-BI function was associated with altered platelet function and decreased adrenal steroidogenesis. (Funded by the European Community and others.).

ABCA1 mutation carriers with low high-density lipoprotein cholesterol are characterized by a larger atherosclerotic burden.

AIMS: Low HDL-C is a potent risk factor for cardiovascular disease (CVD). Yet, mutations in ABCA1, a major determinant of circulating HDL-C levels, were previously not associated with CVD risk in cohort studies. To study the consequences of low plasma levels of high-density lipoprotein cholesterol (HDL-C) due to ATP-binding cassette transporter A1 (ABCA1) dysfunction for atherosclerotic vascular disease in the carotid arteries. METHODS AND RESULTS: We performed 3.0 Tesla magnetic resonance imaging (MRI) measurements of the carotid arteries in 36 carriers of high impact functional ABCA1 mutations and 36 normolipidemic controls. Carriers presented with 42% lower HDL-C levels (P < 0.001), a larger mean wall area (18.6 ± 6.0 vs. 15.8 ± 4.3 mm(2); P = 0.02), a larger mean wall thickness (0.82 ± 0.21 vs. 0.70 ± 0.14 mm; P = 0.005), and a higher normalized wall index (0.37 ± 0.06 vs. 0.33 ± 0.04; P = 0.005) compared with controls, retaining significance after adjustment for smoking, alcohol consumption, systolic blood pressure, diabetes, body mass index, history of CVD, LDL-C, and statin use (P = 0.002). CONCLUSION: Carriers of loss of function ABCA1 mutations display a larger atherosclerotic burden compared with age and sex-matched controls, implying a higher risk for CVD. Further studies are needed to elucidate the full function of ABCA1 in the protection against atherosclerosis. These data support the development of strategies to up-regulate ABCA1 in patients with established CVD.

Identification of a loss-of-function inducible degrader of the low-density lipoprotein receptor variant in individuals with low circulating low-density lipoprotein.

AIMS: Recent genome-wide association studies suggest that IDOL (also known as MYLIP) contributes to variation in circulating levels of low-density lipoprotein cholesterol (LDL-C). IDOL, an E3-ubiquitin ligase, is a recently identified post-transcriptional regulator of LDLR abundance. Briefly, IDOL promotes degradation of the LDLR thereby limiting LDL uptake. Yet the exact role of IDOL in human lipoprotein metabolism is unclear. Therefore, this study aimed at identifying and functionally characterizing IDOL variants in the Dutch population and to assess their contribution to circulating levels of LDL-C. METHODS AND RESULTS: We sequenced the IDOL coding region in 677 individuals with LDL-C above the 95th percentile adjusted for age and gender (high-LDL-C cohort) in which no mutations in the LDLR, APOB, and PCSK9 could be identified. In addition, IDOL was sequenced in 560 individuals with baseline LDL-C levels below the 20th percentile adjusted for age and gender (low-LDL-C cohort). We identified a total of 14 IDOL variants (5 synonymous, 8 non-synonymous, and 1 non-sense). Functional characterization of these variants demonstrated that the p.Arg266X variant represents a complete loss of IDOL function unable to promote ubiquitylation and subsequent degradation of the LDLR. Consistent with loss of IDOL function, this variant was identified in individuals with low circulating LDL-C. CONCLUSION: Our results support the notion that IDOL contributes to variation in circulating levels of LDL-C. Strategies to inhibit IDOL activity may therefore provide a novel therapeutic venue to treating dyslipidaemia.