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

Infusion of C-labeled metabolites provides a gold standard for understanding the metabolic processes used by T cells during immune responses in vivo. Through infusion of C-labeled metabolites (glucose, glutamine, and acetate) in -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.

C 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 . Through infusion of 13C-labeled metabolites (glucose, glutamine, acetate) in ()-infected mice, we demonstrate that CD8+ T effector (Teff) cells utilize metabolites for specific pathways during specific phases of activation. Highly proliferative early Teff cells shunt glucose primarily towards nucleotide synthesis and leverage glutamine anaplerosis in the tricarboxylic acid (TCA) cycle to support ATP and pyrimidine synthesis.

Glutaminase inhibition impairs CD8 T cell activation in STK11-/Lkb1-deficient lung cancer.

The tumor microenvironment (TME) contains a rich source of nutrients that sustains cell growth and facilitate tumor development. Glucose and glutamine in the TME are essential for the development and activation of effector T cells that exert antitumor function. Immunotherapy unleashes T cell antitumor function, and although many solid tumors respond well, a significant proportion of patients do not benefit.

Itaconate confers tolerance to late NLRP3 inflammasome activation.

Itaconate is a unique regulatory metabolite that is induced upon Toll-like receptor (TLR) stimulation in myeloid cells. Here, we demonstrate major inflammatory tolerance and cell death phenotypes associated with itaconate production in activated macrophages. We show that endogenous itaconate is a key regulator of the signal 2 of NLR family pyrin domain containing 3 (NLRP3) inflammasome activation after long lipopolysaccharide (LPS) priming, which establishes tolerance to late NLRP3 inflammasome activation.

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.

Comparative evaluation of itaconate and its derivatives reveals divergent inflammasome and type I interferon regulation in macrophages.

Following activation, macrophages undergo extensive metabolic rewiring. Production of itaconate through the inducible enzyme IRG1 is a key hallmark of this process. Itaconate inhibits succinate dehydrogenase, has electrophilic properties and is associated with a change in cytokine production.

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.

Methionine Metabolism Shapes T Helper Cell Responses through Regulation of Epigenetic Reprogramming.

Epigenetic modifications on DNA and histones regulate gene expression by modulating chromatin accessibility to transcription machinery. Here we identify methionine as a key nutrient affecting epigenetic reprogramming in CD4 T helper (Th) cells. Using metabolomics, we showed that methionine is rapidly taken up by activated T cells and serves as the major substrate for biosynthesis of the universal methyl donor S-adenosyl-L-methionine (SAM).

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 C-glucose infusion techniques to investigate the metabolism of CD8 T cells responding to Listeria infection.

A chemical biology screen identifies a vulnerability of neuroendocrine cancer cells to SQLE inhibition.

Aberrant metabolism of cancer cells is well appreciated, but the identification of cancer subsets with specific metabolic vulnerabilities remains challenging. We conducted a chemical biology screen and identified a subset of neuroendocrine tumors displaying a striking pattern of sensitivity to inhibition of the cholesterol biosynthetic pathway enzyme squalene epoxidase (SQLE). Using a variety of orthogonal approaches, we demonstrate that sensitivity to SQLE inhibition results not from cholesterol biosynthesis pathway inhibition, but rather surprisingly from the specific and toxic accumulation of the SQLE substrate, squalene.

Relation of plasma ceramides to visceral adiposity, insulin resistance and the development of type 2 diabetes mellitus: the Dallas Heart Study.

Aims/hypothesis: Ceramides are sphingolipids that contribute to insulin resistance in preclinical studies. We hypothesised that plasma ceramides would be associated with body fat distribution, insulin resistance and incident type 2 diabetes in a multi-ethnic cohort.

Methods: A total of 1557 participants in the Dallas Heart Study without type 2 diabetes underwent measurements of metabolic biomarkers, fat depots by MRI and plasma ceramides by liquid chromatography-mass spectrometry.

DGAT2 Inhibition Alters Aspects of Triglyceride Metabolism in Rodents but Not in Non-human Primates.

Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step in triglyceride (TG) synthesis and has been shown to play a role in regulating hepatic very-low-density lipoprotein (VLDL) production in rodents. To explore the potential of DGAT2 as a therapeutic target for the treatment of dyslipidemia, we tested the effects of small-molecule inhibitors and gene silencing both in vitro and in vivo. Consistent with prior reports, chronic inhibition of DGAT2 in a murine model of obesity led to correction of multiple lipid parameters.

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 marked by the production of substantial amounts of itaconate, which has recently been described as an immunoregulatory metabolite . Itaconate and its membrane-permeable derivative dimethyl itaconate (DI) selectively inhibit a subset of cytokines , including IL-6 and IL-12 but not TNF.

Using [H]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 [H]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.

CETP (Cholesteryl Ester Transfer Protein) Inhibition With Anacetrapib Decreases Production of Lipoprotein(a) in Mildly Hypercholesterolemic Subjects.

Objective: Lp(a) [lipoprotein (a)] is composed of apoB (apolipoprotein B) and apo(a) [apolipoprotein (a)] and is an independent risk factor for cardiovascular disease and aortic stenosis. In clinical trials, anacetrapib, a CETP (cholesteryl ester transfer protein) inhibitor, causes significant reductions in plasma Lp(a) levels. We conducted an exploratory study to examine the mechanism for Lp(a) lowering by anacetrapib.

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.

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.

Potential mechanism of enhanced postprandial glucagon-like peptide-1 release following treatment with a diacylglycerol acyltransferase 1 inhibitor.

Studies have demonstrated that blockade of diacylglycerol acyltransferase 1 (DGAT1) leads to prolonged release of glucagon-like peptide 1 (GLP-1) after meal challenge. The current study was undertaken to investigate the mechanism of action underlying the elevated levels of GLP-1 release following pharmacological inhibition of DGAT1. We utilized a potent, specific DGAT1 inhibitor, compound , to investigate the changes in intestinal lipid profile in a mouse model after oral administration of the compound and challenge with tracer containing fatty meal.

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 APOL1‑HDL particles could be causal for disease and might serve as a useful risk stratification marker.

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.

Discovery of Novel Allosteric Non-Bisphosphonate Inhibitors of Farnesyl Pyrophosphate Synthase by Integrated Lead Finding.

Farnesyl pyrophosphate synthase (FPPS) is an established target for the treatment of bone diseases, but also shows promise as an anticancer and anti-infective drug target. Currently available anti-FPPS drugs are active-site-directed bisphosphonate inhibitors, the peculiar pharmacological profile of which is inadequate for therapeutic indications beyond bone diseases. The recent discovery of an allosteric binding site has paved the way toward the development of novel non-bisphosphonate FPPS inhibitors with broader therapeutic potential, notably as immunomodulators in oncology.

Effect of Error Propagation in Stable Isotope Tracer Studies: An Approach for Estimating Impact on Apparent Biochemical Flux.

Stable isotope tracers are widely used to quantify metabolic rates, and yet a limited number of studies have considered the impact of analytical error on estimates of flux. For example, when estimating the contribution of de novo lipogenesis, one typically measures a minimum of four isotope ratios, i.e.

Inhibition of cholesteryl ester transfer protein increases cholesteryl ester content of large HDL independently of HDL-to-HDL homotypic transfer: in vitro vs in vivo comparison using anacetrapib and dalcetrapib.

The increase in high density lipoprotein (HDL)-cholesterol observed with cholesteryl ester transfer protein (CETP) inhibition is commonly attributed to blockade of cholesteryl ester (CE) transfer from HDL to low density lipoprotein particles. In vitro, it has been observed that CETP can mediate transfer of CE between HDL particles ("homotypic transfer"), and it is postulated that this contributes to HDL remodeling and generation of anti-atherogenic pre-beta HDL. Inhibition of CETP could limit this beneficial remodeling and reduce pre-beta HDL levels.

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).