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David G McLaren,
Helene L Cardasis,
Steven S Stout,
Sheng-Ping Wang,
Vivienne Mendoza,
Jose M Castro-Perez,
Paul L Miller,
Beth A Murphy,
Anne Marie Cumiskey,
Michele Cleary, Douglas G Johns,
Stephen F Previs,
Thomas P Roddy
[show abstract]
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ABSTRACT: We have previously reported on a liquid chromatography-mass spectrometry method to determine the disposition of [13C18]-oleic acid following intravenous and oral administration in vivo. This approach has enabled us to study a variety of aspects of lipid metabolism including a quantitative assessment of triglyceride synthesis. Here we present a more rigorous evaluation of the constraints imposed upon the analytical method in order to generate accurate data using this stable-isotope tracer approach along with more detail on relevant analytical figures of merit including limits of quantitation, precision and accuracy. The use of mass isotopomer distribution analysis (MIDA) to quantify plasma triglyceride synthesis is specifically highlighted and a re-evaluation of the underlying mathematics has enabled us to present a simplified series of equations. The derivation of this MIDA model and the significance of all underlying assumptions are explored in detail and examples are given of how it can successfully be applied to detect differences in plasma triglyceride synthesis in lean and high-fat diet fed mouse models. More work is necessary to evaluate the applicability of this approach to triglyceride stores with slower rates of turnover such as in adipose or muscle tissue, however the present report provides investigators with the tools necessary to conduct such studies.
Analytical Chemistry 05/2013; · 5.86 Impact Factor
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Seongah Han,
Lauretta Levoci,
Paul Fisher,
Sheng-Ping Wang,
Karen Gagen,
Ying Chen,
Dan Xie,
Timothy Fisher,
Anka G Ehrhardt,
Andrea M Peier, Douglas G Johns
[show abstract]
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ABSTRACT: Cholesteryl ester transfer protein (CETP) is a target of therapeutic intervention for coronary heart disease. Anacetrapib, a potent inhibitor of CETP, has been shown to reduce LDL-cholesterol by 40% and increase HDL-cholesterol by 140% in patients, and is currently being evaluated in a phase III cardiovascular outcomes trial. HDL is known to possess anti-inflammatory properties, however with such large increases in HDL-cholesterol, it is unclear whether CETP inhibition perturbs HDL functionality such as anti-inflammatory effects on endothelial cells. The purpose of the present study was to determine whether CETP inhibition by anacetrapib affects the anti-inflammatory properties of HDL. HDL was isolated from either hamsters treated with vehicle or anacetrapib for 2weeks, or from normal human subjects treated either placebo, 20mg, or 150mg anacetrapib daily for 2weeks. Anacetrapib treatment increased plasma HDL cholesterol levels by 65% and between 48-82% in hamsters and humans, respectively. Pre-incubation of human aortic endothelial cells with HDL isolated from both control and anacetrapib treated suppressed TNFα induced expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin. Similar results were obtained with human HDL samples pre and post treatment with placebo or anacetrapib. Further, HDL inhibited TNFα-induced MCP-1 secretion, monocyte adhesion and NF-κB activation in endothelial cells, and the inhibition was similar between control and anacetrapib treated groups. These studies demonstrate that anacetrapib treatment does not impair the ability of HDL to suppress an inflammatory response in endothelial cells.
Biochimica et Biophysica Acta 12/2012; · 4.66 Impact Factor
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David G McLaren,
Sheng-Ping Wang,
Steven J Stout,
Dan Xie,
Paul L Miller,
Vivienne Mendoza,
Raymond Rosa,
Jose Castro-Perez,
Stephen F Previs, Douglas G Johns,
Thomas P Roddy
[show abstract]
[hide abstract]
ABSTRACT: Isotopic tracers have been used to examine lipid trafficking for many years, data from those studies have typically yielded novel insight regarding the pathophysiology of dyslipidemia. Previous experimental designs were suitable for studies in humans since relatively large volumes of plasma could be regularly sampled. We have expanded on the earlier logic by applying high-throughput analytical methods that require reduced sample volumes. Specifically, we have examined the possibility of coupling gel-based separations of lipoproteins (e.g. Lipoprint) with LC-MS/MS analyses of complex lipid mixtures as a way to routinely measure the labeling profiles of distinct lipids in discrete lipoprotein subfractions. We demonstrate the ability to measure the incorporation of [U-13C]oleate into triglycerides, phospholipids and cholesterol esters in VLDL, LDL and HDL particles in mice. Although rodent models of dyslipidemia are inherently different from humans because of alterations in enzyme activities and underlying metabolism, rodent models can be used to screen novel compounds for efficacy in altering a given biochemical pathway and therein enable studies of target engagement in vivo. We expect that it is possible to translate our approach for application in other systems, including studies in humans.
The Journal of Lipid Research 10/2012; · 5.56 Impact Factor
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[show abstract]
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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.
Drugs 03/2012; 72(4):491-507. · 4.23 Impact Factor
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Haihong Zhou,
Wenyu Li,
Sheng-Ping Wang,
Vivienne Mendoza,
Raymond Rosa,
James Hubert,
Kithsiri Herath,
Theresa McLaughlin,
Rory J Rohm,
Michael E Lassman,
Kenny K Wong, Douglas G Johns,
Stephen F Previs,
Brian K Hubbard,
Thomas P Roddy
[show abstract]
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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. · 5.56 Impact Factor
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James P Conway, Douglas G Johns,
Sheng-Ping Wang,
Nykia D Walker,
Thomas A McAvoy,
Haihong Zhou,
Xuemei Zhao,
Stephen F Previs,
Thomas P Roddy,
Brian K Hubbard,
Nathan A Yates,
Ronald C Hendrickson
[show abstract]
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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. · 5.11 Impact Factor
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02/2012; , ISBN: 978-953-51-0070-6
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A Phillip Owens,
Freda H Passam,
Silvio Antoniak,
Stephanie M Marshall,
Allison L McDaniel,
Lawrence Rudel,
Julie C Williams,
Brian K Hubbard,
Julie-Ann Dutton,
Jianguo Wang, [......],
Alan Daugherty,
Daniel Kirchhofer,
James P Luyendyk,
Patrick M Moriarty,
Shanmugam Nagarajan,
Barbara C Furie,
Bruce Furie, Douglas G Johns,
Ryan E Temel,
Nigel Mackman
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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
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Liwen Zhang,
Timothy McCabe,
Jon H Condra,
Yan G Ni,
Laurence B Peterson,
Weirong Wang,
Alison M Strack,
Fubao Wang,
Shilpa Pandit,
Holly Hammond, [......],
Jayne Chin,
Lyndon J Mitnaul,
Andrea Pellacani,
Carl P Sparrow,
Zhiqiang An,
William Strohl,
Brian Hubbard,
Andrew S Plump,
Daniel Blom,
Ayesha Sitlani
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ABSTRACT: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a promising therapeutic target for treating coronary heart disease. We report a novel antibody 1B20 that binds to PCSK9 with sub-nanomolar affinity and antagonizes PCSK9 function in-vitro. In CETP/LDLR-hemi mice two successive doses of 1B20, administered 14 days apart at 3 or 10 mpk, induced dose dependent reductions in LDL-cholesterol (≥ 25% for 7-14 days) that correlated well with the extent of PCSK9 occupancy by the antibody. In addition, 1B20 induces increases in total plasma antibody-bound PCSK9 levels and decreases in liver mRNA levels of SREBP-regulated genes PCSK9 and LDLR, with a time course that parallels decreases in plasma LDL-cholesterol (LDL-C). Consistent with this observation in mice, in statin-responsive human primary hepatocytes, 1B20 lowers PCSK9 and LDLR mRNA levels and raises serum steady-state levels of antibody-bound PCSK9. In addition, mRNA levels of several SREBP regulated genes involved in cholesterol and fatty-acid synthesis including ACSS2, FDPS, IDI1, MVD, HMGCR, and CYP51A1 were decreased significantly with antibody treatment of primary human hepatocytes. In rhesus monkeys, subcutaneous (SC) dosing of 1B20 dose-dependently induces robust LDL-C lowering (maximal ~70%), which is correlated with increases in target engagement and total antibody-bound PCSK9 levels. Importantly, a combination of 1B20 and Simvastatin in dyslipidemic rhesus monkeys reduced LDL-C more than either agent alone, consistent with a mechanism of action that predicts additive effects of anti-PCSK9 agents with statins. Our results suggest that antibodies targeting PCSK9 could provide patients powerful LDL lowering efficacy on top of statins, and lower cardiovascular risk.
International journal of biological sciences 01/2012; 8(3):310-27. · 2.70 Impact Factor
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Oscar Puig,
Jeffrey Yuan,
Sergey Stepaniants,
Renata Zieba,
Emanuel Zycband,
Mark Morris,
Silvija Coulter,
Xiang Yu,
John Menke,
John Woods, [......],
Xuanmin He,
Edward A O'Neill,
Eric Hailman, Douglas G Johns,
Brian K Hubbard,
Pek Yee Lum,
Samuel D Wright,
Mary M Desouza,
Andrew Plump,
Vladimír Reiser
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ABSTRACT: Atherosclerosis is a complex disease requiring improvements in diagnostic techniques and therapeutic treatments. Both improvements will be facilitated by greater exploration of the biology of atherosclerotic plaque. To this end, we carried out large-scale gene expression analysis of human atherosclerotic lesions.
Whole genome expression analysis of 101 plaques from patients with peripheral artery disease identified a robust gene signature (1514 genes) that is dominated by processes related to Toll-like receptor signaling, T-cell activation, cholesterol efflux, oxidative stress response, inflammatory cytokine production, vasoconstriction, and lysosomal activity. Further analysis of gene expression in microdissected carotid plaque samples revealed that this signature is differentially expressed in macrophage-rich and smooth muscle cell-containing regions. A quantitative PCR gene expression panel and inflammatory composite score were developed on the basis of the atherosclerotic plaque gene signature. When applied to serial sections of carotid plaque, the inflammatory composite score was observed to correlate with histological and morphological features related to plaque vulnerability.
The robust mRNA expression signature identified in the present report is associated with pathological features of vulnerable atherosclerotic plaque and may be useful as a source of biomarkers and targets of novel antiatherosclerotic therapies.
Circulation Cardiovascular Genetics 12/2011; 4(6):595-604. · 6.11 Impact Factor
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Zhu Chen,
Edward A O'Neill,
Roger D Meurer,
Karen Gagen,
Silvi Luell,
Sheng-Ping Wang,
Marina Ichetovkin,
Betsy Frantz-Wattley,
Suzanne Eveland,
Alison M Strack,
Timothy S Fisher, Douglas G Johns,
Carl P Sparrow,
Samuel D Wright,
Brian K Hubbard,
Ester Carballo-Jane
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ABSTRACT: High-density lipoprotein (HDL)-targeting therapies, including reconstituted HDL (rHDL), are attractive agents for treating dyslipidemia and atherosclerosis, as they may increase HDL levels and enhance therapeutic activities associated with HDL, including reverse cholesterol transport (RCT). Using CSL-111, a rHDL consisting of native human apolipoprotein AI (hApoAI) and phospholipids, we characterized the acute effects of rHDL administration in C57Bl/6 mice to (i) further our understanding of the mechanism of action of rHDL, and (ii) evaluate the usefulness of the mouse as a preclinical model for HDL-targeting therapies. After a single injection of CSL-111, there was a dose- and time-dependent increase of hApoAI, human pre-β HDL, total cholesterol, and triglycerides in serum, consistent with the effects of CSL-111 in humans. However, unlike in humans, there was no measurable increase in cholesteryl esters. Evaluated ex vivo, the ATP binding cassette A1 (ABCA1)- and scavenger receptor type BI (SR-BI)-dependent cholesterol efflux capacity of serum from CSL-111-treated mice was increased compared with serum from vehicle-treated animals. Fractionation by size exclusion chromatography of lipoproteins in serum from treated mice revealed hApoAI in particles the size of endogenous HDL and slightly larger, cholesterol-enriched particles of all sizes, including sizes distinct from endogenous HDL or CSL-111 itself, and triglyceride-enriched particles the size of very-low-density lipoprotein (VLDL). These results suggest that in mouse blood CSL-111 is remodeled and generates enhanced cholesterol efflux capacity which increases mobilization of free cholesterol from peripheral tissues. Our findings complement the previous reports on CSL-111 in human participants and provide data with which to evaluate the potential utility of mouse models in mechanistic studies of HDL-targeting therapies.
Journal of Cardiovascular Pharmacology and Therapeutics 11/2011; 17(3):315-23. · 1.75 Impact Factor
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Kristian K Jensen,
Stephen F Previs,
Lei Zhu,
Kithsiri Herath,
Sheng-Ping Wang,
Gowri Bhat,
Guanghui Hu,
Paul L Miller,
David G McLaren,
Myung K Shin,
Thomas F Vogt,
Liangsu Wang,
Kenny K Wong,
Thomas P Roddy, Douglas G Johns,
Brian K Hubbard
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ABSTRACT: The liver is a crossroad for metabolism of lipid and carbohydrates, with acetyl-CoA serving as an important metabolic intermediate and a precursor for fatty acid and cholesterol biosynthesis pathways. A better understanding of the regulation of these pathways requires an experimental approach that provides both quantitative metabolic flux measurements and mechanistic insight. Under conditions of high carbohydrate availability, excess carbon is converted into free fatty acids and triglyceride for storage, but it is not clear how excessive carbohydrate availability affects cholesterol biosynthesis. To address this, C57BL/6J mice were fed either a low-fat, high-carbohydrate diet or a high-fat, carbohydrate-free diet. At the end of the dietary intervention, the two groups received (2)H(2)O to trace de novo fatty acid and cholesterol synthesis, and livers were collected for gene expression analysis. Expression of lipid and glucose metabolism genes was determined using a custom-designed pathway focused PCR-based gene expression array. The expression analysis showed downregulation of cholesterol biosynthesis genes and upregulation of fatty acid synthesis genes in mice receiving the high-carbohydrate diet compared with the carbohydrate-free diet. In support of these findings, (2)H(2)O tracer data showed that fatty acid synthesis was increased 10-fold and cholesterol synthesis was reduced by 1.6-fold in mice fed the respective diets. In conclusion, by applying gene expression analysis and tracer methodology, we show that fatty acid and cholesterol synthesis are differentially regulated when the carbohydrate intake in mice is altered.
AJP Endocrinology and Metabolism 11/2011; 302(2):E209-17. · 4.75 Impact Factor
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Zhu Chen,
Sheng-ping Wang,
Mihajlo L Krsmanovic,
Jose Castro-Perez,
Karen Gagen,
Vivienne Mendoza,
Ray Rosa,
Vinit Shah,
Timothy He,
Steve J Stout,
Neil S Geoghagen,
Sang H Lee,
David G McLaren,
Liangsu Wang,
Thomas P Roddy,
Andrew S Plump,
Brian K Hubbard,
Christopher J Sinz, Douglas G Johns
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ABSTRACT: The objective was to assess whether pharmacological activation of lecithin cholesterol acyltransferase (LCAT) could exert beneficial effects on lipoprotein metabolism. A putative small molecule activator (compound A) was used as a tool compound in in vitro and in vivo studies. Compound A increased LCAT activity in vitro in plasma from mouse, hamster, rhesus monkey, and human. To assess the acute pharmacodynamic effects of compound A, C57Bl/6 mice and hamsters received a single dose (20 mg/kg) of compound A. Both species displayed a significant increase in high-density lipoprotein cholesterol (HDLc) and a significant decrease in non-HDLc and triglycerides acutely after dosing; these changes tracked with ex vivo plasma LCAT activity. To examine compound A's chronic effect on lipoprotein metabolism, hamsters received a daily dosing of vehicle or of 20 or 60 mg/kg of compound A for 2 weeks. At study termination, compound treatment resulted in a significant increase in HDLc, HDL particle size, plasma apolipoprotein A-I level, and plasma cholesteryl ester (CE) to free cholesterol ratio, and a significant reduction in very low-density lipoprotein cholesterol. The increase in plasma CE mirrored the increase in HDL CE. Triglycerides trended toward a dose-dependent decrease in very low-density lipoprotein and HDL, with multiple triglyceride species reaching statistical significance. Gallbladder bile acids content displayed a significant and more than 2-fold increase with the 60 mg/kg treatment. We characterized pharmacological activation of LCAT by a small molecule extensively for the first time, and our findings support the potential of this approach in treating dyslipidemia and atherosclerosis; our analyses also provide mechanistic insight on LCAT's role in lipoprotein metabolism.
Metabolism: clinical and experimental 10/2011; 61(4):470-81. · 2.59 Impact Factor
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Jose Castro-Perez,
Thomas P Roddy,
Nico M M Nibbering,
Vinit Shah,
David G McLaren,
Stephen Previs,
Athula B Attygalle,
Kithsiri Herath,
Zhu Chen,
Sheng-Ping Wang,
Lyndon Mitnaul,
Brian K Hubbard,
Rob J Vreeken, Douglas G Johns,
Thomas Hankemeier
Journal of the American Society for Mass Spectrometry 09/2011; 22(9):1568-9. · 4.00 Impact Factor
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Jose Castro-Perez,
Thomas P Roddy,
Nico M M Nibbering,
Vinit Shah,
David G McLaren,
Stephen Previs,
Athula B Attygalle,
Kithsiri Herath,
Zhu Chen,
Sheng-Ping Wang,
Lyndon Mitnaul,
Brian K Hubbard,
Rob J Vreeken, Douglas G Johns,
Thomas Hankemeier
[show abstract]
[hide abstract]
ABSTRACT: A high content molecular fragmentation for the analysis of phosphatidylcholines (PC) was achieved utilizing a two-stage [trap (first generation fragmentation) and transfer (second generation fragmentation)] collision-induced dissociation (CID) in combination with travelling-wave ion mobility spectrometry (TWIMS). The novel aspects of this work reside in the fact that a TWIMS arrangement was used to obtain a high level structural information including location of fatty acyl substituents and double bonds for PCs in plasma, and the presence of alkali metal adduct ions such as [M + Li](+) was not required to obtain double bond positions. Elemental compositions for fragment ions were confirmed by accurate mass measurements. A very specific first generation fragment ion m/z 577 (M-phosphoryl choline) from the PC [16:0/18:1 (9Z)] was produced, which by further CID generated acylium ions containing either the fatty acyl 16:0 (C(15)H(31)CO(+), m/z 239) or 18:1 (9Z) (C(17)H(33)CO(+), m/z 265) substituent. Subsequent water loss from these acylium ions was key in producing hydrocarbon fragment ions mainly from the α-proximal position of the carbonyl group such as the hydrocarbon ion m/z 67 (+H(2)C-HC = CH-CH = CH(2)). Formation of these ions was of important significance for determining double bonds in the fatty acyl chains. In addition to this, and with the aid of (13)C labeled lyso-phosphatidylcholine (LPC) 18:1 (9Z) in the ω-position (methyl) TAP fragmentation produced the ion at m/z 57. And was proven to be derived from the α-proximal (carboxylate) or distant ω-position (methyl) in the LPC.
Journal of the American Society for Mass Spectrometry 09/2011; 22(9):1552-67. · 4.00 Impact Factor
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Kithsiri Herath,
Gowi Bhat,
Paul L Miller,
Sheng-Ping Wang,
Alison Kulick,
Genevieve Andrews-Kelly,
Christopher Johnson,
Rory J Rohm,
Michael E Lassman,
Stephen F Previs, Douglas G Johns,
Brian K Hubbard,
Thomas P Roddy
[show abstract]
[hide abstract]
ABSTRACT: Protein synthesis can be estimated by measuring the incorporation of a labeled amino acid into a proteolytic peptide. Although prelabeled amino acids are typically administered, recent studies have tested (2)H(2)O; the assumption is that there is rapid equilibration of (2)H (in body water) with the carbon-bound hydrogens of amino acids before those amino acids are incorporated into a protein(s). We have determined the temporal changes in (2)H labeling of body water and amino acids which should build confidence in (2)H(2)O-based studies of protein synthesis when one aims to measure the (2)H labeling of proteolytic peptides.
Analytical Biochemistry 08/2011; 415(2):197-9. · 3.00 Impact Factor
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Jose Castro-Perez,
François Briand,
Karen Gagen,
Sheng-Ping Wang,
Ying Chen,
David G McLaren,
Vinit Shah,
Rob J Vreeken,
Thomas Hankemeier,
Thierry Sulpice,
Thomas P Roddy,
Brian K Hubbard, Douglas G Johns
[show abstract]
[hide abstract]
ABSTRACT: Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester (CE) and triglyceride between HDL and apoB-containing lipoproteins. Anacetrapib (ANA), a reversible inhibitor of CETP, raises HDL cholesterol (HDL-C) and lowers LDL cholesterol in dyslipidemic patients; however, the effects of ANA on cholesterol/lipoprotein metabolism in a dyslipidemic hamster model have not been demonstrated. To test whether ANA (60 mg/kg/day, 2 weeks) promoted reverse cholesterol transport (RCT), ³H-cholesterol-loaded macrophages were injected and (3)H-tracer levels were measured in HDL, liver, and feces. Compared to controls, ANA inhibited CETP (94%) and increased HDL-C (47%). ³H-tracer in HDL increased by 69% in hamsters treated with ANA, suggesting increased cholesterol efflux from macrophages to HDL. ³H-tracer in fecal cholesterol and bile acids increased by 90% and 57%, respectively, indicating increased macrophage-to-feces RCT. Mass spectrometry analysis of HDL from ANA-treated hamsters revealed an increase in free unlabeled cholesterol and CE. Furthermore, bulk cholesterol and cholic acid were increased in feces from ANA-treated hamsters. Using two independent approaches to assess cholesterol metabolism, the current study demonstrates that CETP inhibition with ANA promotes macrophage-to-feces RCT and results in increased fecal cholesterol/bile acid excretion, further supporting its development as a novel lipid therapy for the treatment of dyslipidemia and atherosclerotic vascular disease.
The Journal of Lipid Research 08/2011; 52(11):1965-73. · 5.56 Impact Factor
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Jose M Castro-Perez,
Thomas P Roddy,
Vinit Shah,
Sheng-Ping Wang,
Xuesong Ouyang,
Anthony Ogawa,
David G McLaren,
Marija Tadin-Strapps,
Michael J Robinson,
Steven R Bartz,
Brandon Ason,
Ying Chen,
Stephen F Previs,
Kenny K Wong,
Rob J Vreeken, Douglas G Johns,
Brian K Hubbard,
Thomas Hankemeier,
Lyndon Mitnaul
[show abstract]
[hide abstract]
ABSTRACT: The purpose of this study was to evaluate the use of high resolution LC-MS together with metabolomics and D(4)-cholic acid (D(4)-CA) as a metabolic tracer to measure the metabolism and reconjugation of bile acids (BAs) in vitro and in vivo. Metabolic tracers are very important because they allow for the direct detection (substrate-to-product) of small and significant biological perturbations that may not be apparent when monitoring "static" endogenous levels of particular metabolites. Slc27a5, also known as fatty acid transport protein 5 (FATP5), is the hepatic BA-CoA ligase involved in reconjugating BAs during enterohepatic BA recycling. Using Slc27a5-cKD mice, silencing of ∼90% gene expression was achieved followed by reduction in the reconjugation of D(4)-CA to D(4)-taurocholic acid (D(4)-TCA), as well as other conjugated BA metabolites in plasma (p = 0.0031). The method described allowed a rapid measure of many D(4) and endogenous BA. Analysis of bile resulted in the detection of 39 BA metabolites from a 13 min analytical run. Finally, the utilization of a novel high resolution mass spectrometry method in combination with metabolomics and a stable isotope metabolic tracer allowed for the detection of targeted and untargeted BAs following silencing of the Slc27a5 gene in primary hepatocytes and in mice.
Journal of Proteome Research 08/2011; 10(10):4683-91. · 5.11 Impact Factor
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Jose M Castro-Perez,
Thomas P Roddy,
Vinit Shah,
David G McLaren,
Sheng-Ping Wang,
Kristian Jensen,
Rob J Vreeken,
Thomas Hankemeier, Douglas G Johns,
Stephen F Previs,
Brian K Hubbard
[show abstract]
[hide abstract]
ABSTRACT: A novel method to differentiate diet-induced alterations in plasma lipid phenotypes "static (concentration of lipids) and kinetic (endogenous production, e.g., denovo lipogenesis)" was employed. C57Bl6 mice were randomized into 2 groups and fed either a high-carbohydrate, low-fat (HC) or a carbohydrate-free, high-fat diet (HF) diet for 13 days; D(2)O was administered via intraperitoneal injection and then adding D(2)O to the drinking water for 96 h. Principal component analysis (PCA) revealed differences in the plasma lipid content, for example, triglycerides (TG) 50:2, 50:3, and 52:2 were up-regulated in mice fed the HC diet, whereas TG 52:4, 52:1, 54:5, 54:3, 54:4, and 54:2 were higher in animals fed the HF diet. However, although the fractional contribution of synthesis was ~10-fold lower in HF vs HC fed mice, changes in TG concentration were not entirely mediated by altered de novo lipogenesis. In addition, the ability to couple isotope labeling measurements with PCA analyses revealed cases where there were no differences in the concentration of a compound but its source was substantially altered. In summary, this strategy determined (i) the presence/absence of differences in concentration and (ii) the contribution of different pathways and synthesis that could affect lipid biology in a mouse model respectively.
Journal of Proteome Research 08/2011; 10(9):4281-90. · 5.11 Impact Factor
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Stephen F Previs,
Ablatt Mahsut,
Alison Kulick,
Keiana Dunn,
Genevieve Andrews-Kelly,
Christopher Johnson,
Gowri Bhat,
Kithsiri Herath,
Paul L Miller,
Sheng-Ping Wang,
Karim Azer,
Jing Xu, Douglas G Johns,
Brian K Hubbard,
Thomas P Roddy
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ABSTRACT: The advantages of using (2)H(2)O to quantify cholesterol synthesis include i) homogeneous precursor labeling, ii) incorporation of (2)H via multiple pathways, and iii) the ability to perform long-term studies in free-living subjects. However, there are two concerns. First, the t(1/2) of tracer in body water presents a challenge when there is a need to acutely replicate measurements in the same subject. Second, assumptions are made regarding the number of hydrogens (n) that are incorporated during de novo synthesis. Our primary objective was to determine whether a step-based approach could be used to repeatedly study cholesterol synthesis a subject. We observed comparable changes in the (2)H-labeling of plasma water and total plasma cholesterol in African-Green monkeys that received five oral doses of (2)H(2)O, each dose separated by one week. Similar rates of cholesterol synthesis were estimated when comparing data in the group over the different weeks, but better reproducibility was observed when comparing replicate determinations of cholesterol synthesis in the same nonhuman primate during the respective dosing periods. Our secondary objective was to determine whether n depends on nutritional status in vivo; we observed n of ∼25 and ∼27 in mice fed a high-carbohydrate (HC) versus carbohydrate-free (CF) diet, respectively. We conclude that it is possible to acutely repeat studies of cholesterol synthesis using (2)H(2)O and that n is relatively constant.
The Journal of Lipid Research 07/2011; 52(7):1420-8. · 5.56 Impact Factor
