Metabolic Profiling Using Stable Isotope Tracing Reveals Distinct Patterns of Glucose Utilization by Physiologically Activated CD8+ T Cells.

Naive CD8+ T cells differentiating into effector T cells increase glucose uptake and shift from quiescent to anabolic metabolism. Although much is known about the metabolism of cultured T cells, how T cells use nutrients during immune responses in vivo is less well defined. Here, we combined bioenergetic profiling and 13C-glucose infusion techniques to investigate the metabolism of CD8+ T cells responding to Listeria infection. In contrast to in vitro-activated T cells, which display hallmarks of Warburg metabolism, physiologically activated CD8+ T cells displayed greater rates of oxidative metabolism, higher bioenergetic capacity, differential use of pyruvate, and prominent flow of 13C-glucose carbon to anabolic pathways, including nucleotide and serine biosynthesis. Glucose-dependent serine biosynthesis mediated by the enzyme Phgdh was essential for CD8+ T cell expansion in vivo. Our data highlight fundamental differences in glucose use by pathogen-specific T cells in vivo, illustrating the impact of environment on T cell metabolic phenotypes.

Electrophilic properties of itaconate and derivatives regulate the IκBζ-ATF3 inflammatory axis.

Metabolic regulation has been recognized as a powerful principle guiding immune responses. Inflammatory macrophages undergo extensive metabolic rewiring 1 marked by the production of substantial amounts of itaconate, which has recently been described as an immunoregulatory metabolite 2 . Itaconate and its membrane-permeable derivative dimethyl itaconate (DI) selectively inhibit a subset of cytokines 2 , including IL-6 and IL-12 but not TNF. The major effects of itaconate on cellular metabolism during macrophage activation have been attributed to the inhibition of succinate dehydrogenase2,3, yet this inhibition alone is not sufficient to account for the pronounced immunoregulatory effects observed in the case of DI. Furthermore, the regulatory pathway responsible for such selective effects of itaconate and DI on the inflammatory program has not been defined. Here we show that itaconate and DI induce electrophilic stress, react with glutathione and subsequently induce both Nrf2 (also known as NFE2L2)-dependent and -independent responses. We find that electrophilic stress can selectively regulate secondary, but not primary, transcriptional responses to toll-like receptor stimulation via inhibition of IκBζ protein induction. The regulation of IκBζ is independent of Nrf2, and we identify ATF3 as its key mediator. The inhibitory effect is conserved across species and cell types, and the in vivo administration of DI can ameliorate IL-17-IκBζ-driven skin pathology in a mouse model of psoriasis, highlighting the therapeutic potential of this regulatory pathway. Our results demonstrate that targeting the DI-IκBζ regulatory axis could be an important new strategy for the treatment of IL-17-IκBζ-mediated autoimmune diseases.

Rapid, Selective, and Sensitive Method for Semitargeted Discovery of Congeneric Natural Products by Liquid Chromatography Tandem Mass Spectrometry.

Natural product congeners serve a useful role in the understanding of natural product biosynthesis and structure-activity relationships. A minor congener with superior activity, selectivity, and modifiable functional groups could serve as a more effective lead structure and replace even the original lead molecule that was used for medicinal chemistry modifications. Currently, no effective method exists to discover targeted congeners rapidly, specifically, and selectively from producing sources. Herein, a new method based on liquid-chromatography tandem-mass spectrometry combination is evaluated for targeted discovery of congeners of platensimycin and platencin from the extracts of Streptomyces platensis. By utilizing a precursor-ion searching protocol, tandem mass spectrometry not only confirmed the presence of known congeners but also provided unambiguous detection of many previously unknown congeners of platensimycin and platencin. This high-throughput and quantitative method can be rapidly and broadly applied for dereplication and congener discovery from a variety of producing sources, even when the targeted compounds are obscured by the presence of unrelated natural products.

Preclinical toxicology profile of squalene epoxidase inhibitors.

Small cell lung cancer (SCLC) is a particularly aggressive subset of lung cancer, and identification of new therapeutic options is of significant interest. We recently reported that SCLC cell lines display a specific vulnerability to inhibition of squalene epoxidase (SQLE), an enzyme in the cholesterol biosynthetic pathway that catalyzes the conversion of squalene to 2,3-oxidosqualene. Since it has been reported that SQLE inhibition can result in dermatitis in dogs, we conducted a series of experiments to determine if SQLE inhibitors would be tolerated at exposures predicted to drive maximal efficacy in SCLC tumors. Detailed profiling of the SQLE inhibitor NB-598 showed that dogs did not tolerate predicted efficacious exposures, with dose-limiting toxicity due to gastrointestinal clinical observations, although skin toxicities were also observed. To extend these studies, two SQLE inhibitors, NB-598 and Cmpd-4″, and their structurally inactive analogs, NB-598.ia and Cmpd-4″.ia, were profiled in monkeys. While both active SQLE inhibitors resulted in dose-limiting gastrointestinal toxicity, the structurally similar inactive analogs did not. Collectively, our data demonstrate that significant toxicities arise at exposures well below the predicted levels needed for anti-tumor activity. The on-target nature of the toxicities identified is likely to limit the potential therapeutic utility of SQLE inhibition for the treatment of SCLC.

Using [2H]water to quantify the contribution of de novo palmitate synthesis in plasma: enabling back-to-back studies.

An increased contribution of de novo lipogenesis (DNL) may play a role in cases of dyslipidemia and adipose accretion; this suggests that inhibition of fatty acid synthesis may affect clinical phenotypes. Since it is not clear whether modulation of one step in the lipogenic pathway is more important than another, the use of tracer methods can provide a deeper level of insight regarding the control of metabolic activity. Although [2H]water is generally considered a reliable tracer for quantifying DNL in vivo (it yields a homogenous and quantifiable precursor labeling), the relatively long half-life of body water is thought to limit the ability of performing repeat studies in the same subjects; this can create a bottleneck in the development and evaluation of novel therapeutics for inhibiting DNL. Herein, we demonstrate the ability to perform back-to-back studies of DNL using [2H]water. However, this work uncovered special circumstances that affect the data interpretation, i.e., it is possible to obtain seemingly negative values for DNL. Using a rodent model, we have identified a physiological mechanism that explains the data. We show that one can use [2H]water to test inhibitors of DNL by performing back-to-back studies in higher species [i.e., treat nonhuman primates with platensimycin, an inhibitor of fatty acid synthase]; studies also demonstrate the unsuitability of [13C]acetate.

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.

Plasma Levels of Risk-Variant APOL1 Do Not Associate with Renal Disease in a Population-Based Cohort.

Two common missense variants in APOL1 (G1 and G2) have been definitively linked to CKD in black Americans. However, not all individuals with the renal-risk genotype develop CKD, and little is known about how APOL1 variants drive disease. Given the association of APOL1 with HDL particles, which are cleared by the kidney, differences in the level or quality of mutant APOL1­HDL particles could be causal for disease and might serve as a useful risk stratification marker. We measured plasma levels of G0 (low risk), G1, and G2 APOL1 in 3450 individuals in the Dallas Heart Study using a liquid chromatography-MS method that enabled quantitation of the different variants. Additionally, we characterized native APOL1­HDL from donors with no or two APOL1 risk alleles by size-exclusion chromatography and analysis of immunopurified APOL1­HDL particles. Finally, we identified genetic loci associated with plasma APOL1 levels and tested for APOL1-dependent association with renal function. Although we replicated the previous association between APOL1 variant status and renal function in nondiabetic individuals, levels of circulating APOL1 did not associate with microalbuminuria or GFR. Furthermore, the size or known components of APOL1­HDL did not consistently differ in subjects with the renal-risk genotype. Genetic association studies implicated variants in loci harboring haptoglobin-related protein (HPR), APOL1, and ubiquitin D (UBD) in the regulation of plasma APOL1 levels, but these variants did not associate with renal function. Collectively, these data demonstrate that the risk of renal disease associated with APOL1 is probably not related to circulating levels of the mutant protein.

Evaluation of CETP activity in vivo under non-steady-state conditions: influence of anacetrapib on HDL-TG flux.

Studies in lipoprotein kinetics almost exclusively rely on steady-state approaches to modeling. Herein, we have used a non-steady-state experimental design to examine the role of cholesteryl ester transfer protein (CETP) in mediating HDL-TG flux in vivo in rhesus macaques, and therefore, we developed an alternative strategy to model the data. Two isotopomers ([(2)H11] and [(13)C18]) of oleic acid were administered (orally and intravenously, respectively) to serve as precursors for labeling TGs in apoB-containing lipoproteins. The flux of a specific TG (52:2) from these donor lipoproteins to HDL was used as the measure of CETP activity; calculations are also presented to estimate total HDL-TG flux. Based on our data, we estimate that the peak total postprandial TG flux to HDL via CETP is ∼ 13 mg · h(-1) · kg(-1) and show that this transfer was inhibited by 97% following anacetrapib treatment. Collectively, these data demonstrate that HDL TG flux can be used as a measure of CETP activity in vivo. The fact that the donor lipoproteins can be labeled in situ using well-established stable isotope tracer techniques suggests ways to measure this activity for native lipoproteins in free-living subjects under any physiological conditions.

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.

The hepatitis C virus core protein inhibits adipose triglyceride lipase (ATGL)-mediated lipid mobilization and enhances the ATGL interaction with comparative gene identification 58 (CGI-58) and lipid droplets.

Liver steatosis is a common health problem associated with hepatitis C virus (HCV) and an important risk factor for the development of liver fibrosis and cancer. Steatosis is caused by triglycerides (TG) accumulating in lipid droplets (LDs), cellular organelles composed of neutral lipids surrounded by a monolayer of phospholipids. The HCV nucleocapsid core localizes to the surface of LDs and induces steatosis in cultured cells and mouse livers by decreasing intracellular TG degradation (lipolysis). Here we report that core at the surface of LDs interferes with the activity of adipose triglyceride lipase (ATGL), the key lipolytic enzyme in the first step of TG breakdown. Expressing core in livers or mouse embryonic fibroblasts of ATGL(-/-) mice no longer decreases TG degradation as observed in LDs from wild-type mice, supporting the model that core reduces lipolysis by engaging ATGL. Core must localize at LDs to inhibit lipolysis, as ex vivo TG hydrolysis is impaired in purified LDs coated with core but not when free core is added to LDs. Coimmunoprecipitation experiments revealed that core does not directly interact with the ATGL complex but, unexpectedly, increased the interaction between ATGL and its activator CGI-58 as well as the recruitment of both proteins to LDs. These data link the anti-lipolytic activity of the HCV core protein with altered ATGL binding to CGI-58 and the enhanced association of both proteins with LDs.

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.

Static and turnover kinetic measurement of protein biomarkers involved in triglyceride metabolism including apoB48 and apoA5 by LC/MS/MS.

LC/MS quantification of multiple plasma proteins that differ by several orders of magnitude in concentration from a single sample is challenging. We present a strategy that allows the simultaneous determination of the concentration and turnover kinetics of higher and lower abundant proteins from a single digestion mixture. Our attention was directed at a cluster of proteins that interact to affect the absorption and interorgan lipid trafficking. We demonstrate that apos involved in TG metabolism such as apoC2, C3, E, and A4 (micromolar concentration), and apoB48 and apoA5 (single-digit nanomolar concentration) can be quantified from a single digestion mixture. A high degree of correlation between LC/MS and immunobased measurements for apoC2, C3, E, and B48 was observed. Moreover, apoA5 fractional synthesis rate was measured in humans for the first time. Finally, the method can be directly applied to studies involving nonhuman primates because peptide sequences used in the method are conserved between humans and nonhuman primates.

Simultaneous quantitation and size characterization of apolipoprotein(a) by ultra-performance liquid chromatography/mass spectrometry.

RATIONALE: Apolipoprotein(a) is a polymorphic glycoprotein covalently bound to apoB100 in Lp(a) particles and has been described to be both atherogenic and prothrombotic, although its exact mechanism of action is not well defined. Apolipoprotein(a) is routinely measured by immunoassays. Unfortunately, the accuracy of the measurement can be affected by the apolipoprotein(a) size (number of kringles) polymorphism in Lp(a) particles. Here we describe an ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) assay that is capable of measuring apolipoprotein(a) concentrations while simultaneously determining the number of kringles present per protein. METHODS: Plasma samples were diluted and proteins de-lipidated with deoxycholate prior to tryptic digestion. Distinct tryptic peptides from different regions of apolipoprotein(a) were measured to determine both concentration and the number of kringles present per protein. Separation and quantitation of tryptic peptides is carried out at 700 µL/min using a 1.7 µm C18 column (2.1 × 100 mm) coupled to a Thermo Vantage triple quadrupole (QQQ) mass spectrometer with a heated electrospray ionization (HESI) source. RESULTS: This method was compared to established methods for measuring concentration (monoclonal antibody based ELISA) and size (gel-electrophoresis) using 80 plasma samples proved by NWLRL. The slope and r(2) value for the correlation of concentrations were determined to be 0.96 and 0.98, demonstrating excellent agreement of absolute values between the UPLC/MS and ELISA methods. As measured by UPLC/MS, the average kringle number or size is smaller than determined by the electrophoretic method. CONCLUSIONS: A single UPLC/MS method was developed capable of measuring apolipoprotein(a) concentration and size (by measuring the number of kringles per protein). This assay passes criteria required for 'fit for purpose' assays including sensitivity, intra and interday reproducibility and freeze/thaw stability. While the agreement between UPLC/MS and ELISA is excellent for concentration and may provide researchers with additional tools for studying apolipoprotein(a), the dissimilarities between UPLC/MS and the electrophoretic method may also be exploited for understanding apolipoprotein(a) structure and function.

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.

Determination of low levels of 2H-labeling using high-resolution mass spectrometry: application in studies of lipid flux and beyond.

RATIONALE: The ability to measure low levels of (2)H-labeling is important in studies of metabolic flux, e.g. one can estimate lipid synthesis by administering (2)H2O and then measuring the incorporation of (2)H into fatty acids. Unfortunately, the analyses are complicated by the presence of more abundant naturally occurring stable isotopes, e.g. (13)C. Conventional approaches rely on coupling gas chromatographic separation of lipids with either quadrupole-mass spectrometry (q-MS) and/or pyrolysis-isotope ratio mass spectrometry (IRMS). The former is limited by high background labeling (primarily from (13)C) whereas the latter is not suitable for routine high-throughput analyses. METHODS: We have contrasted the use of continuous flow-pyrolysis-IRMS against high-resolution mass spectrometry (i.e. Qq-FT-ICR MS) for measuring the (2)H-enrichment of fatty acids and peptides. RESULTS: In contrast to IRMS, which requires ~30 min per analysis, it is possible to measure the (2)H-enrichment of palmitate via direct infusion high-resolution mass spectrometry (HRMS) in ~3 min per sample. In addition, Qq-FT-ICR MS enabled measurements of the (2)H-enrichment of peptides (which is not possible using IRMS). CONCLUSIONS: High-resolution mass spectrometry can be used to measure low levels of (2)H-labeling so we expect that this approach will enhance studies of metabolic flux that rely on (2)H-labeled tracers, e.g. (2)H2O. However, since the high-resolution analyses require greater amounts of a given analyte one potential limitation centers on the overall sensitivity. Presumably, future advances can overcome this barrier.

13C metabolite tracing reveals glutamine and acetate as critical in vivo fuels for CD8 T cells.

Infusion of 13C-labeled metabolites provides a gold standard for understanding the metabolic processes used by T cells during immune responses in vivo. Through infusion of 13C-labeled metabolites (glucose, glutamine, and acetate) in Listeria monocytogenes-infected mice, we demonstrate that CD8 T effector (Teff) cells use metabolites for specific pathways during specific phases of activation. Highly proliferative early Teff cells in vivo shunt glucose primarily toward nucleotide synthesis and leverage glutamine anaplerosis in the tricarboxylic acid (TCA) cycle to support adenosine triphosphate and de novo pyrimidine synthesis. In addition, early Teff cells rely on glutamic-oxaloacetic transaminase 1 (Got1)-which regulates de novo aspartate synthesis-for effector cell expansion in vivo. CD8 Teff cells change fuel preference over the course of infection, switching from glutamine- to acetate-dependent TCA cycle metabolism late in infection. This study provides insights into the dynamics of Teff metabolism, illuminating distinct pathways of fuel consumption associated with CD8 Teff cell function in vivo.

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.

Effects of small interfering RNA-mediated hepatic glucagon receptor inhibition on lipid metabolism in db/db mice.

Hepatic glucose overproduction is a major characteristic of type 2 diabetes. Because 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 LDL fraction, and no change in VLDL HDL fractions. Further studies showed that the increase in LDL was the result of 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.

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.