[Show abstract][Hide abstract] ABSTRACT: While genetic determinants strongly influence high density lipoprotein cholesterol (HDLc) levels, most genetic causes underlying variation in HDLc remain unknown. We aimed to identify novel, rare mutations with large effects in candidate genes contributing to extreme HDLc in humans, utilizing family based Mendelian genetics. We performed next-generation sequencing of 456 candidate HDLc-regulating genes in 200 unrelated probands with extremely low (≤10th percentile) or high (≥90th percentile) HDLc. Probands were excluded if known mutations existed in the established HDLc-regulating genes ABCA1, APOA1, LCAT, CETP, LIPG, and GALNT2. We identified 93 novel coding or splice-site variants in 72 candidate genes. Each variant was genotyped in the probands family. Family-based association analyses were performed for variants with sufficient power to detect significance at p<0.05 with a total of 627 family members being assessed. Mutations in the genes GCKR, RNASEL, LILRA3, and DNAH10 segregated with elevated HDLc levels in families, while no mutations associated with low HDLc. Taken together, we have identified mutations in 4 novel genes that may play a role in regulating HDLc levels in humans.
[Show abstract][Hide abstract] ABSTRACT: We discovered a series of novel and potent thiazolylpyridinone-based SCD1 inhibitors based on a 2-aminothiazole HTS hit by replacing the amide bond with a pyridinone moiety. Compound 19 demonstrated good potency against SCD1 in vitro and in vivo. The mouse liver microsomal SCD1 in vitro potency for 19 was improved by more than 240-fold compared to the original HTS hit. Furthermore, 19 demonstrated a dose-dependent reduction of plasma desaturation index with an ED50 of 6.3mg/kg. Compound 19 demonstrated high liver to plasma and liver to eyelid exposures, indicating preferential liver distribution. The preliminary toxicology study with compound 19 did not demonstrate adverse effects related to SCD1 inhibition, suggesting a wide safety margin with respect to other known SCD1 inhibitors with wider distribution profiles.
[Show abstract][Hide abstract] ABSTRACT: Several five- and six-membered heterocycles were introduced to replace the C2-position amide bond of the original 2-aminothiazole-based hit compound 5. Specifically, replacement of the amide bond with an imidazolidinone moiety yielded a novel and potent thiazolylimidazolidinone series of SCD1 inhibitors. XEN723 (compound 22) was identified after optimization of the thiazolylimidazolidinone series. This compound demonstrated a 560-fold improvement in in vitro potency and reduced plasma desaturation indices in a dose dependent manner, with an EC50 of 4.5mg/kg.
[Show abstract][Hide abstract] ABSTRACT: Hepatic glucose overproduction is a major characteristic of type 2 diabetes. Since glucagon is a key regulator for glucose homeostasis, antagonizing the glucagon receptor (Gcgr) is a possible therapeutic strategy for the treatment of diabetes mellitus. To study the effect of hepatic Gcgr inhibition on the regulation of lipid metabolism, we generated siRNA-mediated GCGR knockdown (si-Gcgr) in the db/db mouse. The hepatic knockdown of Gcgr markedly reduced plasma glucose levels; however, total plasma cholesterol was increased. The detailed lipid analysis showed an increase in the low-density lipoprotein fraction, and no change in very-low-density and high-density lipoprotein fractions. Further studies showed that the increase in low-density lipoprotein was the result from over-expression of hepatic lipogenic genes and elevated de novo lipid synthesis. Inhibition of hepatic glucagon signaling via siRNA-mediated Gcgr knockdown had an effect on both glucose and lipid metabolism in db/db mice.
The Journal of Lipid Research 07/2013; · 4.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: -Endothelial lipase is a phospholipase with activity against high density lipoprotein. Although a small number of mutations in LIPG have been described, the role of LIPG in protection against atherosclerosis is unclear. METHODS AND RESULTS: -We identified eight loss-of-function (LOF) mutations in LIPG in individuals with high HDL-C. Functional analysis confirmed that most rare mutations abolish lipase activity in vitro, indicating complete LOF (CLOF), while two more common mutations N396S and R476W reduce activity by ~50%, indicating partial LOF (PLOF), and implying ~50% and ~75% remaining EL function in heterozygous CLOF and PLOF mutation carriers respectively. CLOF mutation carriers had significantly higher plasma HDL-C levels compared to PLOF mutation carriers. Apo-B depleted serum from CLOF carriers showed significantly enhanced cholesterol efflux acceptor capacity, whereas only trends were observed in PLOF carriers. Carriers of LIPG mutations exhibited trends toward reduced CAD in four independent cohorts (meta-analysis OR=0.7, p=0.04). CONCLUSIONS: -Our data suggest that the impact of LIPG mutations is directly related to their effect on EL function, and support that antagonism of EL function improves cardioprotection.
[Show abstract][Hide abstract] ABSTRACT: The use of nicotinic acid to treat dyslipidemia is limited by induction of a "flushing" response, mediated in part by the interaction of prostaglandin D2 (PGD2) with its G-protein coupled receptor DP1 (Ptgdr). The impact of DP1 blockade (genetic or pharmacologic) was assessed in experimental murine models of atherosclerosis. In Ptgdr-/-ApoE-/- mice vs. ApoE-/- mice, both fed a high-fat diet, aortic cholesterol content was modestly higher (1.3- to 1.5-fold, P<0.05) in Ptgdr-/-ApoE-/- mice at 16 and 24 weeks of age, but not at 32 weeks. In multiple ApoE-/- mouse studies, a DP1-specific antagonist L-655 generally had a neutral to beneficial effect on aortic lipids in the presence or absence of nicotinic acid treatment. In a separate study, a modest increase in some atherosclerotic measures was observed with L-655 treatment in Ldlr-/- mice fed a high-fat diet for 8 weeks; however, this effect was not sustained for 16 or 24 weeks. In the same study, treatment with nicotinic acid alone generally decreased plasma and/or aortic lipids, and addition of L-655 did not negate those beneficial effects. These studies demonstrate that inhibition of DP1, with or without nicotinic acid treatment, does not lead to consistent or sustained effects on plaque burden in mouse atherosclerotic models.
The Journal of Lipid Research 10/2012; · 4.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: High DGAT1 expression levels in the small intestine highlight the critical role this enzyme plays in nutrient absorption. Identification of inhibitors which predominantly inhibit DGAT1 in the gut is an attractive drug discovery strategy with anticipated benefits of reduced systemic toxicity. In this report we describe our discovery and optimization of DGAT1 inhibitors whose plasma exposure is minimized by the action of transporters, including the P-glycoprotein transporter. The impact of this unique absorption profile on efficacy in rat and dog efficacy models is presented.
[Show abstract][Hide abstract] ABSTRACT: Lowering of serum low-density lipoprotein cholesterol (LDL-C) levels remains the primary aim of lipid management. Much progress has been made in reducing rates of cardiovascular disease morbidity and mortality, largely through increased awareness of lipid-lowering therapies and particularly through the use of high-efficacy LDL-C-lowering HMG-CoA reductase inhibitors (statins). While statins have been effective in reducing cardiovascular disease risk, many patients do not adequately achieve guideline-recommended LDL-C goals and may benefit from additional cholesterol management therapies. Low serum levels of high-density lipoprotein cholesterol (HDL-C) are considered another important determinant of cardiovascular disease risk, and increased serum HDL-C levels have been shown to be associated with reductions in the incidence of cardiovascular disease. One approach toward raising serum HDL-C levels is the inhibition of cholesteryl ester-transfer protein (CETP), a plasma protein that promotes the transfer of cholesteryl ester from HDL particles and other lipoprotein fractions to pro-atherogenic apolipoprotein B-containing lipoproteins. The inhibition of this protein raises HDL-C levels and also reduces LDL-C levels. The concept of raising HDL-C levels through pharmacological intervention of this target was validated in preclinical and clinical studies with torcetrapib, the first CETP inhibitor to be assessed in late-stage clinical trials. The large clinical outcomes trial, ILLUMINATE, was prematurely terminated due to other unexpected pharmacological effects of torcetrapib that led to an increased risk of cardiovascular events and deaths. Thus, the ultimate effect of CETP inhibition on cardiovascular disease outcomes remains to be determined. Other CETP inhibitors currently in development do not have the adverse effects of increased blood pressure and circulating levels of aldosterone shown to be structurally related to torcetrapib. Preclinical and pharmacology studies have shown that these CETP inhibitors are distinct compared with torcetrapib and lack the features related to its off-target pharmacology. These findings indicate that the off-target activities of torcetrapib are not necessarily class effects of CETP inhibitors. Recent clinical trials have shown that dalcetrapib, anacetrapib and evacetrapib, the most advanced of these compounds in development, effectively raise HDL-C levels and lower LDL-C in the absence of off-target activities. The results of these trials are encouraging within the limits of study size and duration and provide a rationale for conducting further studies, including large clinical outcomes trials to assess whether CETP inhibition can lead to cardioprotective effects. This review summarizes the data supporting the development of CETP inhibitors as HDL-C-raising therapy, including structure-activity relationships and preclinical and clinical pharmacology studies of known CETP inhibitors.
[Show abstract][Hide abstract] ABSTRACT: Stable isotope tracer studies of apoprotein flux in rodent models present difficulties as they require working with small volumes of plasma. We demonstrate the ability to measure apoprotein flux by administering either (2)H- or (18)O-labeled water to mice and then subjecting samples to LC-MS/MS analyses; we were able to simultaneously determine the labeling of several proteolytic peptides representing multiple apoproteins. Consistent with relative differences reported in the literature regarding apoprotein flux in humans, we found that the fractional synthetic rate of apoB is greater than apoA1 in mice. In addition, the method is suitable for quantifying acute changes in protein flux: we observed a stimulation of apoB production in mice following an intravenous injection of Intralipid and a decrease in apoB production in mice treated with an inhibitor of microsomal triglyceride transfer protein. In summary, we demonstrate a high-throughput method for studying apoprotein kinetics in rodent models. Although notable differences exist between lipoprotein profiles that are observed in rodents and humans, we expect that the method reported here has merit in studies of dyslipidemia as i) rodent models can be used to probe target engagement in cases where one aims to modulate apoprotein production and ii) the approach should be adaptable to studies in humans.
The Journal of Lipid Research 03/2012; 53(6):1223-31. · 4.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Intracellular proteins are in a state of flux, continually being degraded into amino acids and resynthesized into new proteins. The rate of this biochemical recycling process varies across proteins and is emerging as an important consideration in drug discovery and development. Here, we developed a triple-stage quadrupole mass spectrometry assay based on product ion measurements at unit resolution and H(2)(18)O stable tracer incorporation to measure relative protein synthesis rates. As proof of concept, we selected to measure the relative in vivo synthesis rate of ApoB100, an apolipoprotein where elevated levels are associated with an increased risk of coronary heart disease, in plasma-isolated very low density lipoprotein (VLDL) and low density lipoprotein (LDL) in a mouse in vivo model. In addition, serial time points were acquired to measure the relative in vivo synthesis rate of mouse LDL ApoB100 in response to vehicle, microsomal triacylglycerol transfer protein (MTP) inhibitor, and site-1 protease inhibitor, two potential therapeutic targets to reduce plasma ApoB100 levels at 2 and 6 h post-tracer-injection. The combination of H(2)(18)O tracer with the triple quadrupole mass spectrometry platform creates an assay that is relatively quick and inexpensive to transfer across different biological model systems, serving as an ideal rapid screening tool for relative protein synthesis in response to treatment.
Journal of Proteome Research 03/2012; 11(3):1591-7. · 5.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Current approaches to measure protein turnover that use stable isotope-labeled tracers via GC-MS are limited to a small number of relatively abundant proteins. We developed a multiplexed liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM) assay to measure protein turnover and compared the fractional synthetic rates (FSRs) for 2 proteins, VLDL apolipoprotein B100 (VLDL apoB100) and HDL apoA-I, measured by both methods. We applied this technique to other proteins for which kinetics are not readily measured with GC-MS.
Subjects were given a primed-constant infusion of [5,5,5-D(3)]-leucine (D(3)-leucine) for 15 h with blood samples collected at selected time points. Apolipoproteins isolated by SDS-PAGE from lipoprotein fractions were analyzed by GC-MS or an LC-SRM assay designed to measure the M+3/M+0 ratio at >1% D(3)-leucine incorporation. We calculated the FSR for each apolipoprotein by curve fitting the tracer incorporation data from each subject.
The LC-SRM method was linear over the range of tracer enrichment values tested and highly correlated with GC-MS (R(2) > 0.9). The FSRs determined from both methods were similar for HDL apoA-I and VLDL apoB100. We were able to apply the LC-SRM approach to determine the tracer enrichment of multiple proteins from a single sample as well as proteins isolated from plasma after immunoprecipitation.
The LC-SRM method provides a new technique for measuring the enrichment of proteins labeled with stable isotopes. LC-SRM is amenable to a multiplexed format to provide a relatively rapid and inexpensive means to measure turnover of multiple proteins simultaneously.
[Show abstract][Hide abstract] ABSTRACT: Microsomal triglyceride transfer protein (Mtp) inhibitors represent a novel therapeutic approach to lower circulating LDL cholesterol, although therapeutic development has been hindered by the observed increase in hepatic triglycerides and liver steatosis following treatment. Here, we used small interfering RNAs (siRNA) targeting Mtp to achieve target-specific silencing to study this phenomenon and to determine to what extent liver steatosis is induced by changes in Mtp expression. We observed that Mtp silencing led to a decrease in many genes involved in hepatic triglyceride synthesis. Given the role of diacylglycerol O-acyltransferase 2 (Dgat2) in regulating hepatic triglyceride synthesis, we then evaluated whether target-specific silencing of both Dgat2 and Mtp were sufficient to attenuate Mtp silencing-induced liver steatosis. We showed that the simultaneous inhibition of Dgat2 and Mtp led to a decrease in plasma cholesterol and a reduction in the accumulation of hepatic triglycerides caused by the inhibition of Mtp. Collectively, these findings provide a proof-of-principle for a triglyceride synthesis/Mtp inhibitor combination and represent a potentially novel approach for therapeutic development in which targeting multiple pathways can achieve the desired response.
The Journal of Lipid Research 02/2012; 53(5):859-67. · 4.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genome-wide association studies (GWAS) are useful in studying the complex pathways underlying diseases such as atherosclerosis; however, additional testing is often necessary to identify the disease causal genes linked to GWAS loci. We used siRNA-mediated gene knockdown in primary human hepatocytes (PHuH) to identify potential GWAS causal genes affecting the hepatic secretion of apolipoprotein B (ApoB), ApoA1, and proprotein convertase subtilisin/kexin type 9.
Candidate causal genes within GWAS loci affecting human plasma levels of total cholesterol, LDL-cholesterol, HDL-cholesterol, and triglycerides were identified from the literature; 191 genes were selected from 74 loci. A functional siRNA screen was performed using PHuH.
Four genes: poly (ADP-ribose) polymerases member 10, haptoglobin, fucosyltransferase 1, and lysophosphatidic acid receptor 2 were identified and confirmed. Knocking down these genes reduced cell-associated and secreted ApoB levels.
Modification of these four genes may affect plasma lipids through modulation of ApoB secretion.
[Show abstract][Hide abstract] ABSTRACT: Apolipoprotein B100 (apoB100) and apolipoprotein A1 (apoA1) are the primary protein components of low density lipoprotein (LDL) and high density lipoprotein (HDL) particles, respectively, and plasma levels of these proteins are associated with risks of cardiovascular disease. Existing apoB100 quantitation methods for animal models have been limited to affinity capture techniques such as enzyme-linked immunosorbent assay (ELISA) and Western blot which require specialized reagents for each species and in many cases are not readily available. Here we demonstrate a single translatable ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) assay that is fast and robust and can be used to measure apolipoprotein concentrations in plasma for six species. When possible, peptide sequences that are conserved across species were identified for this assay. The sample preparation is limited and can be carried out in 96-well microtiter plates and thus allows for multiplexed preparation of samples for analysis of large numbers of samples in a short time frame when combined with UPLC/MS/MS. Separation and quantitation of the tryptic peptides is carried out at 700 μL/min using a 1.7 µm core shell C18 column (2.1 × 50 mm). The chromatography is designed for the analysis of over 100 samples per day, and the UPLC run is less than 10 min. This assay is capable of supporting cardiovascular research by providing a single assay to measure critical biomarkers across multiple species without the need for antibodies, and does so in a high-throughput manner.
Rapid Communications in Mass Spectrometry 01/2012; 26(2):101-8. · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hypercholesterolemia is a major risk factor for atherosclerosis. It also is associated with platelet hyperactivity, which increases morbidity and mortality from cardiovascular disease. However, the mechanisms by which hypercholesterolemia produces a procoagulant state remain undefined. Atherosclerosis is associated with accumulation of oxidized lipoproteins within atherosclerotic lesions. Small quantities of oxidized lipoproteins are also present in the circulation of patients with coronary artery disease. We therefore hypothesized that hypercholesterolemia leads to elevated levels of oxidized LDL (oxLDL) in plasma and that this induces expression of the procoagulant protein tissue factor (TF) in monocytes. In support of this hypothesis, we report here that oxLDL induced TF expression in human monocytic cells and monocytes. In addition, patients with familial hypercholesterolemia had elevated levels of plasma microparticle (MP) TF activity. Furthermore, a high-fat diet induced a time-dependent increase in plasma MP TF activity and activation of coagulation in both LDL receptor-deficient mice and African green monkeys. Genetic deficiency of TF in bone marrow cells reduced coagulation in hypercholesterolemic mice, consistent with a major role for monocyte-derived TF in the activation of coagulation. Similarly, a deficiency of either TLR4 or TLR6 reduced levels of MP TF activity. Simvastatin treatment of hypercholesterolemic mice and monkeys reduced oxLDL, monocyte TF expression, MP TF activity, activation of coagulation, and inflammation, without affecting total cholesterol levels. Our results suggest that the prothrombotic state associated with hypercholesterolemia is caused by oxLDL-mediated induction of TF expression in monocytes via engagement of a TLR4/TLR6 complex.
The Journal of clinical investigation 01/2012; 122(2):558-68. · 15.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In an attempt to understand the applicability of various animal models to dyslipidemia in humans and to identify improved preclinical models for target discovery and validation for dyslipidemia, we measured comprehensive plasma lipid profiles in 24 models. These included five mouse strains, six other nonprimate species, and four nonhuman primate (NHP) species, and both healthy animals and animals with metabolic disorders. Dyslipidemic humans were assessed by the same measures. Plasma lipoprotein profiles, eight major plasma lipid fractions, and FA compositions within these lipid fractions were compared both qualitatively and quantitatively across the species. Given the importance of statins in decreasing plasma low-density lipoprotein cholesterol for treatment of dyslipidemia in humans, the responses of these measures to simvastatin treatment were also assessed for each species and compared with dyslipidemic humans. NHPs, followed by dog, were the models that demonstrated closest overall match to dyslipidemic humans. For the subset of the dyslipidemic population with high plasma triglyceride levels, the data also pointed to hamster and db/db mouse as representative models for practical use in target validation. Most traditional models, including rabbit, Zucker diabetic fatty rat, and the majority of mouse models, did not demonstrate overall similarity to dyslipidemic humans in this study.
The Journal of Lipid Research 01/2012; 53(1):51-65. · 4.39 Impact Factor