The use of stable-isotopically labeled oleic acid to interrogate lipid assembly in vivo: assessing pharmacological effects in preclinical species

Abstract

This article is available online at http://www.jlr.orgUnderstanding the synthesis and disposition of triglycerides, cholesteryl esters, and phospholipids is an important component in developing effective therapies to treat cardiovascular and metabolic disease. The kinetics of synthesis and disposition can be ascertained by introducing a labeled substrate and measuring its incorporation into and decay from bulk lipid pools, which provides useful information on the fl ux of lipid through the relevant synthetic and metabolizing pathways. Such techniques have been used to study the kinetics of synthesis of VLDL triglycerides ( 1-3 ), cholesterol ( 4, 5 ), and phospholipids ( 6 ), as well as many other aspects of lipid disposition. When introducing a labeled substrate into a biological system, the pattern of labeling in downstream metabolites conveys information about the synthesis of the product. By experimentally measuring this labeling pattern and comparing the results to those predicted by combinatorial probability, it is possible to determine kinetic parameters. This technique has been termed mass isotopomer distribution analysis (MIDA); discussions on consideration for use and limits of applicability of the method have been presented previously ( 7,8 ). A central tenet of MIDA is that differences in the observed concentration of a particular isotopomeric product can be due solely to the relative abundances of the labeled precursor and the endogenous form of the substrate, i.e., the labeling of the precursor pool. In effect, introduction of the label represents an intervention on the system, and this carries important ramifi cations for interpreting observed effects when a second Abstract The use of stable isotopically labeled substrates and analysis by mass spectrometry have provided substantial insight into rates of synthesis, disposition, and utilization of lipids in vivo. The information to be gained from such studies is of particular benefi t to therapeutic research where the underlying causes of disease may be related to the production and utilization of lipids. When studying biology through the use of isotope tracers, care must be exercised in interpreting the data to ensure that any response observed can truly be interpreted as biological and not as an artifact of the experimental design or a dilutional effect on the isotope. We studied the effects of dosing route and tracer concentration on the mass isotopomer distribution profi le as well as the action of selective inhibitors of microsomal triglyceride transfer protein (MTP) in mice and diacylglycerol acyltransferase 1 (DGAT1) in nonhuman primates, using a stable-isotopically labeled approach. Subjects were treated with inhibitor and subsequently given a dose of uniformly 13 C-labeled oleic acid. Samples were analyzed using a rapid LC-MS technique, allowing the effects of the intervention on the assembly and disposition of triglycerides, cholesteryl esters, and phospholipids to be determined in a single 3 min run from just 10 l of plasma. Press, March...

The use of stable isotope-labeled glycerol and oleic acid to differentiate the hepatic functions of DGAT1 and -2

“…13 C 18 -oleic acid was recently used to elucidate lipid assembly in vivo by measuring the plasma labeling pattern of TG, phospholipids, and cholesteryl esters ( 16 ). Here, we utilized two potent, selective DGAT2 inhibitors to interrogate the role of DGAT2 in 13 C 18 -oleic acid incorporation into TGs in HepG2 cells.…”

DGAT1 mutation is linked to a congenital diarrheal disorder

“…Mice lacking DGAT1 have normal fat absorption, although absorption is delayed and more fat reaches distal intestinal regions (5). Because of the favorable metabolic phenotype of DGAT1-knockout mice (6), DGAT1 inhibitors have been developed (7)(8)(9) and proven efficacious in animal studies (8,10). Several are being evaluated in clinical trials (11)(12)(13).…”

Tracking fatty acid kinetics in distinct lipoprotein fractions in vivo: a novel high-throughput approach for studying dyslipidemia in rodent models

“…Fortunately, advances in mass spectrometry and other areas now permit high-throughput, high-resolution measurements. For example, we, and others recently demonstrated the ability to measure the incorporation of [ 13 C 18 ]oleate into TG following a single bolus administration of tracer ( 5,12 ). We demonstrated the utilization of that approach in quantifying TG synthesis in vivo.…”

“…For example, we recently demonstrated the ability to quantify TG kinetics using a high-throughput mass spectrometry-based assay, in which attention was directed toward an examination of whether the kinetics of an individual TG(s) would refl ect the overall kinetics of the TG pool ( 5 ). In the current study, we have expanded on our earlier logic by directing attention toward an examination of the temporal labeling of distinct analytes in specifi c lipoprotein subfractions.…”

“…Advances in mass spectrometry and stable isotope tracer methods now permit routine analyses of specifi c analytes ( 2,3 ). For example, whereas previous experiments studied the bulk pool of TG, it is now possible to examine individual species (2)(3)(4)(5)(6).…”