Epidemiological and experimental data implicate branched chain amino acids (BCAAs) in the development of insulin resistance, but the mechanisms underlying this link remain unclear.1–3 Insulin resistance in skeletal muscle stems from excess accumulation of lipid species4, a process that requires blood-borne lipids to first traverse the blood vessel wall. Little is known, however, of how this trans-endothelial transport occurs or is regulated. Here, we leverage PGC-1α, a transcriptional coactivator that regulates broad programs of FA consumption, to identify 3-hydroxy-isobutyrate (3-HIB), a catabolic intermediate of the BCAA valine, as a novel paracrine regulator of trans-endothelial fatty acids (FA) transport. 3-HIB is secreted from muscle cells, activates endothelial FA transport, stimulates muscle FA uptake in vivo, and promotes muscle lipid accumulation and insulin resistance in animals. Conversely, inhibiting the synthesis of 3-HIB in muscle cells blocks the promotion of endothelial FA uptake. 3-HIB levels are elevated in muscle from db/db mice and from subjects with diabetes. These data thus unveil a novel mechanism that regulates trans-endothelial flux of FAs, revealing 3-HIB as a new bioactive signaling metabolite that links the regulation of FA flux to BCAA catabolism and provides a mechanistic explanation for how increased BCAA catabolic flux can cause diabetes.

Summary The intricate connection between the circadian clock and metabolism remains poorly understood. We used high temporal resolution metabolite profiling to explore clock regulation of mouse liver and cell autonomous metabolism. In liver, ~50% of metabolites were circadian, with enrichment of nucleotide, amino acid, and methylation pathways. In U2 OS cells, 28% were circadian, including amino acids and NAD biosynthesis metabolites. Eighteen metabolites oscillated in both systems and a subset of these in primary hepatocytes. These 18 metabolites were enriched in methylation and amino acid pathways. To assess clock-dependence of these rhythms, we used genetic perturbation. BMAL1 knockdown diminished metabolite rhythms, while CRY1 or CRY2 perturbation generally shortened/lengthened rhythms, respectively. Surprisingly, CRY1 knockdown induced 8 h rhythms in amino acid, methylation, and vitamin metabolites, decoupling metabolite from transcriptional rhythms, with potential impact on nutrient sensing in vivo. These results provide the first comprehensive views of circadian liver and cell autonomous metabolism.

The intricate connection between the circadian clock and metabolism remains poorly understood. We used high temporal resolution metabolite profiling to explore clock regulation of mouse liver and cell autonomous metabolism. In liver, ~50% of metabolites were circadian, with enrichment of nucleotide, amino acid, and methylation pathways. In U2 OS cells, 28% were circadian, including amino acids and NAD biosynthesis metabolites. Eighteen metabolites oscillated in both systems and a subset of these in primary hepatocytes. These 18 metabolites were enriched in methylation and amino acid pathways. To assess clock-dependence of these rhythms, we used genetic perturbation. BMAL1 knockdown diminished metabolite rhythms, while CRY1 or CRY2 perturbation generally shortened/lengthened rhythms, respectively. Surprisingly, CRY1 knockdown induced 8 h rhythms in amino acid, methylation, and vitamin metabolites, decoupling metabolite from transcriptional rhythms, with potential impact on nutrient sensing in vivo. These results provide the first comprehensive views of circadian liver and cell autonomous metabolism. Blurb Using high temporal resolution metabolite profiling, XXX et al show that over 50% of liver metabolites are circadian, with a significant overlap of cycling metabolites between mouse and human liver, especially those involved in epigenetic regulation. Coupling of metabolite with transcriptional rhythms is regulated by core clock genes. Krishnaiah et al.

The phospholipase A2 (PLA2)activity of phosphorylated peroxiredoxin 6 (Prdx6) is required for activation of NADPH oxidase (NOX2). We investigated the interaction of Prdx6 with p67(phox) and its effect on NOX2 activity. With the use of specific antibodies, coimmunoprecipitation of p67(phox) and phosphorylated Prdx6 was demonstrated with lysates of mouse pulmonary microvascular endothelial cells (MPMVECs) that were stimulated with angiotensin II; the interaction of p67(phox) with nonphosphorylated Prdx6 was relatively weak. Association of p67(phox) and phosphoPrdx6 in intact MPMVECs after angiotensin II stimulation was demonstrated by proximity ligation assay and was abolished by U0126, a MAP kinase inhibitor. By isothermal titration calorimetry, p67(phox) bound strongly to phosphoPrdx6 but bound poorly to Prdx6; phosphorylated p67(phox) did not bind to either Prdx6 or phosphoPrdx6. PLA2 activity of recombinant phosphoPrdx6 was decreased by >98% in the presence of p67(phox); the calculated dissociation constant (Kd) of the p67(phox): phosphoPrdx6 complex was 65 nM. PLA2 activity (MJ33 sensitive) in cell lysates following angiotensin II treatment of MPMVECs was increased by 85% following knockdown of p67(phox) with siRNA. These data indicate that p67(phox) binds to phosphoPrdx6 and inhibits its PLA2 activity, an interaction that could function to terminate the PLA2-mediated NOX2 activation signal.-Krishnaiah, S. Y., Dodia, C., Feinstein, S. I., and Fisher, A. B. p67(phox) terminates the phospholipase A2-derived signal for activation of NADPH oxidase (NOX2).

Peroxiredoxin 6 (Prdx6) is a bifunctional enzyme with peroxidase and phospholipase A2 (PLA2) activities. This protein participates in the degradation and remodeling of internalized dipalmitoylphosphatidylcholine (DPPC), the major phospholipid component of lung surfactant. We have shown previously that the PLA2 activity of Prdx6 is inhibited by the lung surfactant-associated protein called surfactant protein A (SP-A) through direct protein-protein interaction. Docking of SPA and Prdx6 was modeled using the ZDOCK (zlab.bu.edu) program in order to predict molecular sites for binding of the two proteins. The predicted peptide sequences were evaluated for binding to the opposite protein using isothermal titration calorimetry and circular dichroism measurement followed by determination of the effect of the SP-A peptide on the PLA2 activity of Prdx6. The sequences 195EEEAKKLFPK204.in the Prdx6 helix and 83DEELQTELYEIKHQIL99 in SP-A were identified as the sites for hydrophobic interaction and H+-bonding between the 2 proteins. Treatment of mouse endothelial cells with the SP-A peptide inhibited their recovery from lipid peroxidation associated with oxidative stress indicating inhibition of Prdx6 activity by the peptide in the intact cell.

The nonmetabolizable lysophosphatidylcholine (LysoPC) analogue edelfosine is the prototype of a class of compounds being investigated for their potential as selective chemotherapeutic agents. Edelfosine targets membranes, disturbing cellular homeostasis. Is not clear at this point how membrane alterations are communicated between intracellular compartments leading to growth inhibition and eventual cell death. In the present study, a combined metabolomics/lipidomics approach for the unbiased identification of metabolic pathways altered in yeast treated with sublethal concentrations of the LysoPC analogue was employed. Mass spectrometry of polar metabolites, fatty acids, and lipidomic profiling was used to study the effects of edelfosine on yeast metabolism. Amino acid and sugar metabolism, the Krebs cycle, and fatty acid profiles were most disrupted, with polar metabolites and short-medium chain fatty acid changes preceding long and very long-chain fatty acid variations. Initial increases in metabolites such as trehalose, proline, and γ-amino butyric acid with a concomitant decrease in metabolites of the Krebs cycle, citrate and fumarate, are interpreted as a cellular attempt to offset oxidative stress in response to mitochondrial dysfunction induced by the treatment. Notably, alanine, inositol, and myristoleic acid showed a steady increase during the period analyzed (2, 4, and 6 h after treatment). Of importance was the finding that edelfosine induced significant alterations in neutral glycerolipid metabolism resulting in a significant increase in the signaling lipid diacylglycerol.

Oscillations in circadian metabolism are crucial to the well being of organism. Our understanding of metabolic rhythms has been greatly enhanced by recent advances in high-throughput systems biology experimental techniques and data analysis. In an in vitro setting, metabolite rhythms can be measured by time-dependent sampling over an experimental period spanning one or more days at sufficent resolution to elucidate rhythms. We hypothesized that cellular metabolic effects over such a time course would be influenced by both oscillatory and circadian-independent cell metabolic effects. Here we use nuclear magnetic resonance (NMR) spectroscopy-based metabolic profiling of mammalian cell culture media of synchronized U2 OS cells containing an intact transcriptional clock. The experiment was conducted over 48 h, typical for circadian biology studies, and samples collected at 2 h resolution to unravel such non-oscillatory effects. Our data suggest specific metabolic activities exist that change continuously over time in this settting and we demonstrate that the non-oscillatory effects are generally monotonic and possible to model with multivariate regression. Deconvolution of such non-circadian persistent changes are of paramount importance to consider while studying circadian metabolic oscillations.

The metabolic requirements of metastatic non-small cell lung (mNSCLC) tumors from patients receiving first-line platinum-doublet chemotherapy are hypothesized to imprint a blood signature suitable for survival prediction. Pre-treatment samples prospectively collected at baseline from a randomized phase III trial were assayed using nuclear magnetic resonance (NMR) spectroscopy (n = 341) and ultra-high performance liquid chromatography – mass spectrometry (UPLC-MS) (n = 297). Distributions of time to event outcomes were estimated by Kaplan-Meier analysis, and baseline characteristics adjusted Cox regression modeling was used to correlate markers’ levels to time to event outcomes. Sixteen polar metabolites were significantly correlated with overall survival (OS) by univariate analysis (p < 0.025). Formate, 2-hydroxybutyrate, glycine and myo-inositol were selected for a multivariate model. The median OS was 6.6 months in the high-risk group compared to 11.4 months in the low risk group HR (Hazard Ratio) = 1.99, 95% C.I. (Confidence Interval) 1.45–2.68; p < 0.0001). Modeling of lipids by class (sphingolipids, acylcarnitines and lysophosphatidylcholines) revealed a median OS = 5.7 months vs. 11. 9 months for the high vs. low risk group. (HR: 2.23, 95% C.I. 1.55–3.20; p < 0.0001). These results demonstrate that metabolic profiles from pre-treatment samples may be useful to stratify clinical outcomes for mNSCLC patients receiving chemotherapy. Genomic and longitudinal measurements pre- and post-treatment may yield addition information to personalize treatment decisions further.