Edited by Luke O'NeillItaconic acid is an important metabolite produced by macrophages after stimulation with LPS. the cell culture medium leads to elevated intracellular levels of unlabeled succinate, with no evidence of intracellular uptake. The goal of this study is to encourage the development of effective pro-drug strategies to increase the intracellular levels of itaconate, which will enable more conclusive analysis of its action on macrophages and other cell and tissue types.Itaconic acid is a dicarboxylic acid polar metabolite originally characterized in Aspergillus terreus, but is also produced in mammalian cells (1). After LPS stimulation, itaconic acid is secreted by macrophages, where it can inhibit bacterial cell growth (1). In macrophages, itaconate synthesis is catalyzed by the immune-responsive gene 1 (IRG1) protein, which mediates the decarboxylation of cis-aconitate to itaconate (2). Metabolomic and fluxomic analysis of LPS-stimulated macrophages demonstrated reduced Isocitrate dehydrogenase-1 (IDH1) expression and increased IRG1 expression, resulting in a diversion of citrate from the TCA cycle 2 toward itaconate production (3). This metabolic remodeling results in glutamate serving as the anaplerotic substrate to maintain or elevate succinate levels (3). Moreover, elevated succinate acts as an inflammatory signal that induces secretion of IL-1␤ and stabilization of HIF-1␣ (4). Based on the similarity between itaconate (methylene succinic acid) and succinate, recent investigations have focused on the link between itaconate synthesis and succinate accumulation. Two complementary studies reported that itaconate inhibits succinate dehydrogenase and drives succinate accumulation (5, 6). Studying the role of itaconate requires either lowering its intracellular concentration by using IRG1 knock-out mouse models (5, 6) or increasing its intracellular concentration by using either itaconate (5) or a "cell-permeable" analog, dimethyl itaconate (DMI) (6). However, there is no direct evidence that itaconate or DMI can cross cell membranes and increase intracellular itaconate. Without direct evidence of intracellular delivery, it remains unclear whether itaconate-mediated metabolic and inflammatory effects are induced by increasing intracellular itaconate or by an extracellular mechanism.Here we synthesized isotopically labeled [ 13 C]itaconate and dimethyl [13 C]itaconate ([ 13 C]DMI) to directly profile itaconate metabolism and uptake (Fig. 1). This analysis suggests that exogenous itaconate is not taken up into cells and [13 C]DMI is not metabolized into [13 C]itaconate in bone marrow-derived macrophages. We also report that [13 C]itaconate in the cell culture medium leads to elevated intracellular levels of unlabeled succinate, yet there is no evidence of intracellular uptake. Overall, this study highlights current limitations in intracellular itaconate delivery, and emphasizes the development of effective pro-drug strategies to conclusively define its action on macrophages and other cell and tissue t...

IntroductionRheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation of the joints and the presence of autoantibodies directed against proteins containing the non-standard arginine-derived amino acid citrulline. The protein fibrinogen, which has an essential role in blood clotting, is one of the most prominent citrullinated autoantigens in RA, particularly because it can be found in the inflamed tissue of affected joints. Here, we set out to analyze the presence of citrullinated endogenous peptides in the synovial fluid of RA and arthritic control patients.MethodsEndogenous peptides were isolated from the synovial fluid of RA patients and controls by filtration and solid phase extraction. The peptides were identified and quantified using high-resolution liquid chromatography-mass spectrometry.ResultsOur data reveal that the synovial fluid of RA patients contains soluble endogenous peptides, derived from fibrinogen, containing significant amounts of citrulline residues and, in some cases, also phosphorylated serine. Several citrullinated peptides are found to be more abundantly present in the synovial fluid of RA patients compared to patients suffering from other inflammatory diseases affecting the joints.ConclusionsThe increased presence of citrullinated peptides in RA patients points toward a possible specific role of these peptides in the immune response at the basis of the recognition of citrullinated peptides and proteins by RA patient autoantibodies.

Background: Pathways underlying fatty acid potentiation of glucose-stimulated insulin secretion have not been fully elucidated.Results: In INS-1 cells, fatty acids increase de novo production of glycerolipids and simultaneously increase glucose utilization. GPR40 receptor activation increases these activities. Conclusion: Fatty acids enhance the production of multiple signals supporting glucose-stimulated insulin secretion. Significance: The studies clarify the effects of fatty acids and GPR40 activity in ␤ cell insulin secretion.

Microfluidics is an enabling technology for both cell biology and chemical analysis. We combine these attributes with a microfluidic device for on-line solid-phase extraction (SPE) and mass spectrometry (MS) analysis of secreted metabolites from living cells in culture on the chip. The device was constructed with polydimethylsiloxane (PDMS) and contains a reversibly-sealed chamber for perfusing cells. A multilayer design allowed a series of valves to control an on-chip 7.5 μL injection loop downstream of the cell chamber with operation similar to a 6-port valve. The valve collects sample and then diverts it to a packed SPE bed that was connected in-line to treat samples prior to MS analysis. The valve allows samples to be collected and injected onto the SPE bed while preventing exposure of cells to added back-pressure from the SPE bed and organic solvents needed to elute collected chemicals. Here, cultured murine 3T3-L1 adipocytes were loaded into the cell chamber and non-esterified fatty acids (NEFAs) that were secreted by the cells were monitored by SPE-MS at 30 min intervals. The limit of detection for a palmitoleic acid standard was 1.4 μM. Due to the multiplexed detection capabilities of MS, a variety of NEFAs were detected. Upon stimulation with isoproterenol and forskolin, secretion of select NEFAs was elevated an average of 1.5-fold compared to basal levels. Despite the 30 min delay between sample injections, this device is a step towards a miniaturized system that allows automated monitoring and identification of a variety of molecules in the extracellular environment.

AMPK regulates many metabolic pathways including fatty acid and glucose metabolism, both of which are closely associated with insulin secretion in pancreatic β-cells. Insulin secretion is regulated by metabolic coupling factors such as ATP/ADP ratio and other metabolites generated by the metabolism of nutrients such as glucose, fatty acid and amino acids. However, the connection between AMPK activation and insulin secretion in β-cells has not yet been fully elucidated at a metabolic level. To study the effect of AMPK activation on glucose stimulated insulin secretion, we applied the pharmacological activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to an INS-1 (832/13) β-cell line. We measured the change in 66 metabolites in the presence or absence of AICAR using different stable isotopic labeled nutrients to probe selected pathways. AMPK activation by AICAR increased basal insulin secretion and reduced the glucose stimulation index. Although ATP/ADP ratios were not strongly affected by AICAR, several other metabolites and pathways important for insulin secretion were affected by AICAR treatment including long-chain CoAs, malonyl-CoA, 3-hydroxy-3 methylglutaryl CoA, diacylglycerol, and farnesyl pyrophosphate. Tracer studies using 13C-glucose revealed lower glucose flux in the purine and pyrimidine pathway and in the glycerolipid synthesis pathway. Untargeted metabolomics revealed reduction in ceramides caused by AICAR that may explain the beneficial role of AMPK in protecting β-cells from lipotoxicity. Taken together, the results provide an overall picture of the metabolic changes associated with AICAR treatment and how it modulates insulin secretion and β-cell survival.