Erratum: Non-homeostatic body weight regulation through a brainstem-restricted receptor for GDF15.

This corrects the article DOI: 10.1038/nature24042.

Non-homeostatic body weight regulation through a brainstem-restricted receptor for GDF15.

Under homeostatic conditions, animals use well-defined hypothalamic neural circuits to help maintain stable body weight, by integrating metabolic and hormonal signals from the periphery to balance food consumption and energy expenditure. In stressed or disease conditions, however, animals use alternative neuronal pathways to adapt to the metabolic challenges of altered energy demand. Recent studies have identified brain areas outside the hypothalamus that are activated under these 'non-homeostatic' conditions, but the molecular nature of the peripheral signals and brain-localized receptors that activate these circuits remains elusive. Here we identify glial cell-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) as a brainstem-restricted receptor for growth and differentiation factor 15 (GDF15). GDF15 regulates food intake, energy expenditure and body weight in response to metabolic and toxin-induced stresses; we show that Gfral knockout mice are hyperphagic under stressed conditions and are resistant to chemotherapy-induced anorexia and body weight loss. GDF15 activates GFRAL-expressing neurons localized exclusively in the area postrema and nucleus tractus solitarius of the mouse brainstem. It then triggers the activation of neurons localized within the parabrachial nucleus and central amygdala, which constitute part of the 'emergency circuit' that shapes feeding responses to stressful conditions. GDF15 levels increase in response to tissue stress and injury, and elevated levels are associated with body weight loss in numerous chronic human diseases. By isolating GFRAL as the receptor for GDF15-induced anorexia and weight loss, we identify a mechanistic basis for the non-homeostatic regulation of neural circuitry by a peripheral signal associated with tissue damage and stress. These findings provide opportunities to develop therapeutic agents for the treatment of disorders with altered energy demand.

Trophic Dynamics and Feeding Ecology of Skipjack Tuna (Katsuwonus pelamis) off Eastern and Western Taiwan.

The skipjack tuna (Katsuwonus pelamis) is a mesopredator fish species with seasonal abundance in waters off Taiwan. Regional ecological and life-history information has been historically lacking for this species. In recent years, stable isotope analysis (SIA) of carbon and nitrogen has been used to assess predator feeding ecology and broader ecosystem trophic dynamics. This study evaluated comparative skipjack feeding ecology in distinct regions off Taiwan, combining traditional stomach content analysis with SIA of individuals off western (n = 43; 2020) and eastern (n = 347; 2012-2014 and n = 167; 2020) Taiwan. The stomach content analysis showed the most important prey to be ponyfish (Photopectoralis bindus) in western Taiwan and epipelagic squids (Myopsina spp.) and carangids (Decapterus macrosoma;) in eastern Taiwan from 2012 to 2014 and epipelagic carangids (Decapterus spp.) and flying fishes (Cheilopogon spp.) in eastern Taiwan in 2020, suggesting that the skipjack tuna is a generalist predator across regions. In contrast, time-integrated diet estimates from Bayesian mixing models indicated the importance of cephalopods and crustaceans as prey, potentially demonstrating more mesopelagic feeding in less productive waters during skipjack migrations outside the study regions. Skipjack off western Taiwan had a slightly higher estimated trophic position than in the waters off eastern Taiwan, potentially driven by the varying nutrient-driven pelagic food web structures. Skipjack SI values increased with body size off eastern Taiwan but not in western waters, suggesting that opportunistic predation can still result in different predator-prey size dynamics between regions.

Characterization of Anacetrapib Distribution into the Lipid Droplet of Adipose Tissue in Mice and Human Cultured Adipocytes.

Anacetrapib is an inhibitor of cholesteryl ester transfer protein (CETP), associated with reduction in LDL cholesterol and increase in HDL cholesterol in hypercholesterolemic patients. Anacetrapib was not taken forward into filing/registration as a new drug for coronary artery diease, despite the observation of a ∼9% reduction in cardiovascular risk in a large phase III cardiovascular outcomes trial (REVEAL). Anacetrapib displayed no adverse effects throughout extensive preclinical safety evaluation, and no major safety signals were observed in clinical trials studying anacetrapib, including REVEAL. However, anacetrapib demonstrated a long terminal half-life in all species, thought to be due, in part, to distribution into adipose tissue. We sought to understand the dependence of anacetrapib's long half-life on adipose tissue and to explore potential mechanisms that might contribute to the phenomenon. In mice, anacetrapib localized primarily to the lipid droplet of adipocytes in white adipose tissue; in vitro, anacetrapib entry into cultured human adipocytes depended on the presence of a mature adipocyte and lipid droplet but did not require active transport. In vivo, the entry of anacetrapib into adipose tissue did not require lipase activity, as the distribution of anacetrapib into adipose was-not affected by systemic lipase inhibition using poloaxamer-407, a systemic lipase inhibitor. The data from these studies support the notion that the entry of anacetrapib into adipose tissue/lipid droplets does not require active transport, nor does it require mobilization or entry of fat into adipose via lipolysis.

Quantifying ceramide kinetics in vivo using stable isotope tracers and LC-MS/MS.

Numerous studies have implicated dyslipidemia as a key factor in mediating insulin resistance. Ceramides have received special attention since their levels are inversely associated with normal insulin signaling and positively associated with factors that are involved in cardiometabolic disease. Despite the growing literature surrounding ceramide biology, there are limited data regarding the activity of ceramide synthesis and turnover in vivo. Herein, we demonstrate the ability to measure ceramide kinetics by coupling the administration of [2H]water with LC-MS/MS analyses. As a "proof-of-concept" we determined the effect of a diet-induced alteration on ceramide flux; studies also examined the effect of myriocin (a known inhibitor of serine palmitoyltransferase, the first step in sphingosine biosynthesis). Our data suggest that one can estimate ceramide synthesis and draw conclusions regarding the source of fatty acids; we discuss caveats in regards to method development in this area.

GPR120 suppresses adipose tissue lipolysis and synergizes with GPR40 in antidiabetic efficacy.

GPR40 and GPR120 are fatty acid sensors that play important roles in glucose and energy homeostasis. GPR40 potentiates glucose-dependent insulin secretion and demonstrated in clinical studies robust glucose lowering in type 2 diabetes. GPR120 improves insulin sensitivity in rodents, albeit its mechanism of action is not fully understood. Here, we postulated that the antidiabetic efficacy of GPR40 could be enhanced by coactivating GPR120. A combination of GPR40 and GPR120 agonists in db/db mice, as well as a single molecule with dual agonist activities, achieved superior glycemic control compared with either monotherapy. Compared with a GPR40 selective agonist, the dual agonist improved insulin sensitivity in ob/ob mice measured by hyperinsulinemic-euglycemic clamp, preserved islet morphology, and increased expression of several key lipolytic genes in adipose tissue of Zucker diabetic fatty rats. Novel insights into the mechanism of action for GPR120 were obtained. Selective GPR120 activation suppressed lipolysis in primary white adipocytes, although this effect was attenuated in adipocytes from obese rats and obese rhesus, and sensitized the antilipolytic effect of insulin in rat and rhesus primary adipocytes. In conclusion, GPR120 agonism enhances insulin action in adipose tissue and yields a synergistic efficacy when combined with GPR40 agonism.

Enhancing Studies of Pharmacodynamic Mechanisms via Measurements of Metabolic Flux: Fundamental Concepts and Guiding Principles for Using Stable Isotope Tracers.

Drug discovery and development efforts are largely based around a common expectation, namely, that direct or indirect action on a cellular process (e.g., statin-mediated enzyme inhibition or insulin-stimulated receptor activation) will have a beneficial impact on physiologic homeostasis. To expand on this, one could argue that virtually all pharmacologic interventions attempt to influence the flow of "traffic" in a biochemical network, irrespective of disease or modality. Since stable isotope tracer kinetic methods provide a measure of traffic flow (i.e., metabolic flux), their inclusion in study designs can yield novel information regarding pathway biology; the application of such methods requires the integration of knowledge in physiology, analytical chemistry, and mathematical modeling. Herein, we review the fundamental concepts that surround the use of tracer kinetics, define basic terms, and outline guiding principles via theoretical and experimental problems. Specifically, one needs to 1) recognize the types of biochemical events that change isotopic enrichments, 2) appreciate the distinction between fractional turnover and flux rate, and 3) be aware of the subtle differences between tracer kinetics and pharmacokinetics. We hope investigators can use the framework presented here to develop applications that address their specific questions surrounding biochemical flux, and thereby gain insight into the pathophysiology of disease states, and examine pharmacodynamic mechanisms.

Dose-dependent effects of siRNA-mediated inhibition of SCAP on PCSK9, LDLR, and plasma lipids in mouse and rhesus monkey.

SREBP cleavage-activating protein (SCAP) is a key protein in the regulation of lipid metabolism and a potential target for treatment of dyslipidemia. SCAP is required for activation of the transcription factors SREBP-1 and -2. SREBPs regulate the expression of genes involved in fatty acid and cholesterol biosynthesis, and LDL-C clearance through the regulation of LDL receptor (LDLR) and PCSK9 expression. To further test the potential of SCAP as a novel target for treatment of dyslipidemia, we used siRNAs to inhibit hepatic SCAP expression and assess the effect on PCSK9, LDLR, and lipids in mice and rhesus monkeys. In mice, robust liver Scap mRNA knockdown (KD) was achieved, accompanied by dose-dependent reduction in SREBP-regulated gene expression, de novo lipogenesis, and plasma PCSK9 and lipids. In rhesus monkeys, over 90% SCAP mRNA KD was achieved resulting in approximately 75, 50, and 50% reduction of plasma PCSK9, TG, and LDL-C, respectively. Inhibition of SCAP function was demonstrated by reduced expression of SREBP-regulated genes and de novo lipogenesis. In conclusion, siRNA-mediated inhibition of SCAP resulted in a significant reduction in circulating PCSK9 and LDL-C in rodent and primate models supporting SCAP as a novel target for the treatment of dyslipidemia.

Quantifying rates of glucose production in vivo following an intraperitoneal tracer bolus.

Aberrant regulation of glucose production makes a critical contribution to the impaired glycemic control that is observed in type 2 diabetes. Although isotopic tracer methods have proven to be informative in quantifying the magnitude of such alterations, it is presumed that one must rely on venous access to administer glucose tracers which therein presents obstacles for the routine application of tracer methods in rodent models. Since intraperitoneal injections are readily used to deliver glucose challenges and/or dose potential therapeutics, we hypothesized that this route could also be used to administer a glucose tracer. The ability to then reliably estimate glucose flux would require attention toward setting a schedule for collecting samples and choosing a distribution volume. For example, glucose production can be calculated by multiplying the fractional turnover rate by the pool size. We have taken a step-wise approach to examine the potential of using an intraperitoneal tracer administration in rat and mouse models. First, we compared the kinetics of [U-13C]glucose following either an intravenous or an intraperitoneal injection. Second, we tested whether the intraperitoneal method could detect a pharmacological manipulation of glucose production. Finally, we contrasted a potential application of the intraperitoneal method against the glucose-insulin clamp. We conclude that it is possible to 1) quantify glucose production using an intraperitoneal injection of tracer and 2) derive a "glucose production index" by coupling estimates of basal glucose production with measurements of fasting insulin concentration; this yields a proxy for clamp-derived assessments of insulin sensitivity of endogenous production.

Assessment of fibrotic tissue and microvascular architecture by in-line phase-contrast imaging in a mouse model of liver fibrosis.

PURPOSE: To explore the value of in-line phase-contrast imaging with computed tomography (ILPCI-CT) by synchrotron radiation (SR) for liver fibrosis. MATERIALS AND METHODS: Liver fibrosis models were set up in 13 BALB/c mice by peritoneal injections of thioacetamide and evaluated by ILPCI-CT. Histological staging was used to categorize liver fibrosis into normal, mild fibrosis and advanced fibrosis groups. Microvessel density (MVD), the ratio of total vessel length to volume (L/V), the ratio of total number of branching points to liver volume (P/V) and the distribution of vessel diameter were assessed. RESULTS: The CT images showed slightly high-density shadows around the portal tracts in the fibrosis group. Three-dimensional reconstruction can detect vascular and nodular changes on the surface of fibrotic livers. The MVDs between the three groups were significantly different (P = 0.024). L/V was significantly different between the three groups (P = 0.014). There was a positive correlation between MVD and P/V. CONCLUSION: Fibrous material can be detected by ILPCI-CT even in the early stage of fibrosis. MVD, L/V, P/V and the distribution of vessel diameter were consistent with fibrosis-related angiogenesis progress. Three-dimensional reconstruction is a promising method to visualize morphological changes of the fibrotic liver. KEY POINTS: • ILPCI-CT can detect fibrous material even in the early stage of liver fibrosis. • MVD, L/V, P/V, and the distribution of vascular diameter reflect pathological angiogenesis. • 3D reconstruction could be a promising approach for detecting liver fibrosis.

Quantitative profiling of oxylipins in plasma and atherosclerotic plaques of hypercholesterolemic rabbits.

Oxylipins are oxidation products of polyunsaturated fatty acids (PUFAs) that affect a broad range of physiological processes, including cell proliferation, inflammation, inflammation resolution, and vascular function. Moreover, oxylipins are readily detectable in plasma, and certain subsets of oxylipins have been detected in human atherosclerotic lesions. Taken together, we set out to produce a detailed quantitative assessment of plasma and plaque oxylipins in a widely used model of atherosclerosis, to identify potential biomarkers of disease progression. We administered regular chow or regular chow supplemented with 0.5% cholesterol (HC) to male New Zealand white rabbits for 12 weeks to induce hypercholesterolemia and atherosclerosis. Our targeted lipidomic analyses of oxylipins on plaques isolated from rabbits fed the HC diet detected 34 oxylipins, 28 of which were in compliance with our previously established quality control acceptance criteria. The arachidonic acid (AA) metabolite derived from the COX pathway, 6-keto-PGF1α was the most abundant plaque oxylipin, followed by the linoleic acid (LA) metabolites 9-HODE, 13-HODE and 9,12,13-TriHOME and the arachidonic acid (AA)-derivatives 11-HETE and 12-HETE. We additionally found that the most abundant oxylipins in plasma were three of the five most abundant oxylipins in plaque, namely 11-HETE, 13-HODE, and 9-HODE. The studies reported here make the first step towards a comprehensive characterization of oxylipins as potentially translatable biomarkers of atherosclerosis.

Glucagon receptor antagonism induces increased cholesterol absorption.

Glucagon and insulin have opposing action in governing glucose homeostasis. In type 2 diabetes mellitus (T2DM), plasma glucagon is characteristically elevated, contributing to increased gluconeogenesis and hyperglycemia. Therefore, glucagon receptor (GCGR) antagonism has been proposed as a pharmacologic approach to treat T2DM. In support of this concept, a potent small-molecule GCGR antagonist (GRA), MK-0893, demonstrated dose-dependent efficacy to reduce hyperglycemia, with an HbA1c reduction of 1.5% at the 80 mg dose for 12 weeks in T2DM. However, GRA treatment was associated with dose-dependent elevation of plasma LDL-cholesterol (LDL-c). The current studies investigated the cause for increased LDL-c. We report findings that link MK-0893 with increased glucagon-like peptide 2 and cholesterol absorption. There was not, however, a GRA-related modulation of cholesterol synthesis. These findings were replicated using structurally diverse GRAs. To examine potential pharmacologic mitigation, coadministration of ezetimibe (a potent inhibitor of cholesterol absorption) in mice abrogated the GRA-associated increase of LDL-c. Although the molecular mechanism is unknown, our results provide a novel finding by which glucagon and, hence, GCGR antagonism govern cholesterol metabolism.

New methodologies for studying lipid synthesis and turnover: looking backwards to enable moving forwards.

Our ability to understand the pathogenesis of problems surrounding lipid accretion requires attention towards quantifying lipid kinetics. In addition, studies of metabolic flux should also help unravel mechanisms that lead to imbalances in inter-organ lipid trafficking which contribute to dyslipidemia and/or peripheral lipid accumulation (e.g. hepatic fat deposits). This review aims to outline the development and use of novel methods for studying lipid kinetics in vivo. Although our focus is directed towards some of the approaches that are currently reported in the literature, we include a discussion of the older literature in order to put "new" methods in better perspective and inform readers of valuable historical research. Presumably, future advances in understanding lipid dynamics will benefit from a careful consideration of the past efforts, where possible we have tried to identify seminal papers or those that provide clear data to emphasize essential points. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease.

Tracer-based estimates of protein flux in cases of incomplete product renewal: evidence and implications of heterogeneity in collagen turnover.

The synthesis of various molecules can be estimated by measuring the incorporation of a labeled precursor into a product of interest. Unfortunately, a central problem in many studies has been an inability to estimate the intracellular dilution of the precursor and therein correctly calculate the synthesis of the product; it is generally assumed that measuring the true product labeling is straightforward. We initiated a study to examine liver collagen synthesis and identified an apparent problem with assumptions regarding measurements of the product labeling. Since it is well known that collagen production is relatively slow, we relied on the use of [(2)H]H2O labeling (analogous to a primed infusion) and sampled animals over the course of 16 days. Although the water labeling (the precursor) remained stable and we observed the incorporation of labeled amino acids into collagen, the asymptotic protein labeling was considerably lower than what would be expected based on the precursor labeling. Although this observation is not necessarily surprising (i.e., one might expect that a substantial fraction of the collagen pool would appear "inert" or turn over at a very slow rate), its implications are of interest in certain areas. Herein, we discuss a novel situation in which tracers are used to quantify rates of flux under conditions where a product may not undergo complete replacement. We demonstrate how heterogeneity in the product pool can lead one to the wrong conclusions regarding estimates of flux, and we outline an approach that may help to minimize errors surrounding data interpretation.

Inhibition of cholesteryl ester transfer protein by anacetrapib does not impair the anti-inflammatory properties of high density lipoprotein.

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 and 82% in hamsters and humans, respectively. Pre-incubation of human aortic endothelial cells with HDL isolated from both control and anacetrapib treated hamsters 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.

In vivo isotopically labeled atherosclerotic aorta plaques in ApoE KO mice and molecular profiling by matrix-assisted laser desorption/ionization mass spectrometric imaging.

RATIONALE: The ability to quantify rates of formation, regression and/or remodeling of atherosclerotic plaque should facilitate a better understanding of the pathogenesis and management of cardiovascular disease. In the current study, we coupled a stable isotope labeled tracer protocol with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to examine spatial and temporal lipid dynamics in atherosclerotic plaque. METHODS: To promote plaque formation in the aorta region, ApoE KO mice were fed a high cholesterol diet (0.15% cholesterol) and orally dosed with (2,2,3,4,4,6-d(6))-cholesterol over several weeks. Tissue sections of ~10 µm thickness were analyzed by MALDI-MSI using matrix deposition by either chemical sublimation or acoustic droplet ejection. RESULTS: MALDI-MSI yielded distinct spatial distribution information for a variety of lipid classes including specific lysophosphatidylcholines typically associated with atherosclerosis-related tissue damage such as phospholipase 2 (Lp-PLA(2)) that mediate chemotactic responses to inflammation (e.g. LPC 16:0, LPC 18:0 and LPC 18:1) as well as free cholesterol and cholesteryl esters that contribute to atheroma formation. MALDI mass spectra acquired from aorta tissue sections clearly distinguished non-esterified and esterified versions of (2,2,3,4,4,6-d(6))-cholesterol within aortic plaque regions and showed distinct spatial accumulation of the cholesterol tracer. CONCLUSIONS: The ability to couple stable isotope based protocols with MALDI-MSI enables a novel strategy to characterize the effects of therapeutic treatments on atherosclerotic plaque formation, regression and potential remodeling of the complex lipid components with high chemical specificity and spatiotemporal information.

In vivo effects of anacetrapib on preß HDL: improvement in HDL remodeling without effects on cholesterol absorption.

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester and triglyceride between HDL and apoB-containing lipoproteins. Anacetrapib (ANA), a reversible inhibitor of CETP, raises HDL cholesterol and lowers LDL cholesterol in dyslipidemic patients. We previously demonstrated that ANA increases macrophage-to-feces reverse cholesterol transport and fecal cholesterol excretion in hamsters, and increased preß HDL-dependent cholesterol efflux via ABCA1 in vitro. However, the effects of ANA on in vivo preß HDL have not been characterized. In vitro, ANA inhibited the formation of preß, however in ANA-treated dyslipidemic hamsters, preß HDL levels (measured by two-dimensional gel electrophoresis) were increased, in contrast to in vitro findings. Because changes in plasma preß HDL have been proposed to potentially affect markers of cholesterol absorption with other CETP inhibitors, a dual stable isotope method was used to directly measure cholesterol absorption in hamsters. ANA treatment of hamsters (on either dyslipidemic or normal diet) had no effect on cholesterol absorption, while dalcetrapib-treated hamsters displayed an increase in cholesterol absorption. Taken together, these data support the notion that ANA promotes preß HDL functionality in vivo, with no effects on cholesterol absorption.

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

Isotopic tracers have been used to examine lipid trafficking for many years, and data from those studies have typically yielded novel insight regarding the pathophysiology of dyslipidemia. Previous experimental designs were suitable for studies in humans because 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-(13)C]oleate into triglycerides (TG), PLs (PL), and cholesterol esters (CE) 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.

Nicotinic acid and DP1 blockade: studies in mouse models of atherosclerosis.

The use of nicotinic acid to treat dyslipidemia is limited by induction of a "flushing" response, mediated in part by the interaction of prostaglandin D(2) (PGD(2)) 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 versus 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.

Use of [13C18] oleic acid and mass isotopomer distribution analysis to study synthesis of plasma triglycerides in vivo: analytical and experimental considerations.

We have previously reported on a liquid chromatography-mass spectrometry method to determine the disposition of [(13)C18]-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.