Phosphoinositide 3-kinase (PI3K) activity is crucial for leukocyte function, but the roles of the four receptor-activated isoforms are unclear. Mice lacking heterotrimeric guanine nucleotide-binding protein (G protein)–coupled PI3Kγ were viable and had fully differentiated neutrophils and macrophages. Chemoattractant-stimulated PI3Kγ ^−/− neutrophils did not produce phosphatidylinositol 3,4,5-trisphosphate, did not activate protein kinase B, and displayed impaired respiratory burst and motility. Peritoneal PI3Kγ-null macrophages showed a reduced migration toward a wide range of chemotactic stimuli and a severely defective accumulation in a septic peritonitis model. These results demonstrate that PI3Kγ is a crucial signaling molecule required for macrophage accumulation in inflammation.

Wortmannin at nanomolar concentrations is a potent and specific inhibitor of phosphoinositide (PI) 3-kinase and has been used extensively to demonstrate the role of this enzyme in diverse signal transduction processes. At higher concentrations, wortmannin inhibits the ataxia telangiectasia gene (ATM)-related DNA-dependent protein kinase (DNA-PKcs). We report here the identification of the site of interaction of wortmannin on the catalytic subunit of PI 3-kinase, p110alpha. At physiological pH (6.5 to 8) wortmannin reacted specifically with p110alpha. Phosphatidylinositol-4,5-diphosphate, ATP, and ATP analogs [adenine and 5'-(4-fluorosulfonylbenzoyl)adenine] competed effectively with wortmannin, while substances containing nucleophilic amino acid side chain functions had no effect at the same concentrations. This suggests that the wortmannin target site is localized in proximity to the substrate-binding site and that residues involved in wortmannin binding have an increased nucleophilicity because of their protein environment. Proteolytic fragments of wortmannin-treated, recombinant p110alpha were mapped with anti-wortmannin and anti-p110alpha peptide antibodies, thus limiting the target site within a 10-kDa fragment, colocalizing with the ATP-binding site. Site-directed mutagenesis of all candidate residues within this region showed that only the conservative Lys-802-to-Arg mutation abolished wortmannin binding. Inhibition of PI 3-kinase occurs, therefore, by the formation of an enamine following the attack of Lys-802 on the furan ring (at C-20) of wortmannin. The Lys-802-to-Arg mutant was also unable to bind FSBA and was catalytically inactive in lipid and protein kinase assays, indicating a crucial role for Lys-802 in the phosphotransfer reaction. In contrast, an Arg-916-to-Pro mutation abolished the catalytic activity whereas covalent wortmannin binding remained intact. Our results provide the basis for the design of novel and specific inhibitors of an enzyme family, including PI kinases and ATM-related genes, that play a central role in many physiological processes.

Glycine is the proteinogenic amino-acid of lowest molecular weight, harboring a hydrogen atom as a side-chain. In addition to being a building-block for proteins, glycine is also required for multiple metabolic pathways, such as glutathione synthesis and regulation of one-carbon metabolism. Although generally viewed as a non-essential amino-acid, because it can be endogenously synthesized to a certain extent, glycine has also been suggested as a conditionally essential amino acid. In metabolic disorders associated with obesity, type 2 diabetes (T2DM), and non-alcoholic fatty liver disease (NAFLDs), lower circulating glycine levels have been consistently observed, and clinical studies suggest the existence of beneficial effects induced by glycine supplementation. The present review aims at synthesizing the recent advances in glycine metabolism, pinpointing its main metabolic pathways, identifying the causes leading to glycine deficiency—especially in obesity and associated metabolic disorders—and evaluating the potential benefits of increasing glycine availability to curb the progression of obesity and obesity-related metabolic disturbances. This study focuses on the importance of diet, gut microbiota, and liver metabolism in determining glycine availability in obesity and associated metabolic disorders.

Butyrate and R-β-hydroxybutyrate are two related short chain fatty acids naturally found in mammals. Butyrate, produced by enteric butyric bacteria, is present at millimolar concentrations in the gastrointestinal tract and at lower levels in blood; R-β-hydroxybutyrate, the main ketone body, produced by the liver during fasting can reach millimolar concentrations in the circulation. Both molecules have been shown to be histone deacetylase (HDAC) inhibitors, and their administration has been associated to an improved metabolic profile and better cellular oxidative status, with butyrate inducing PGC1α and fatty acid oxidation and R-β-hydroxybutyrate upregulating oxidative stress resistance factors FOXO3A and MT2 in mouse kidney. Because of the chemical and functional similarity between the two molecules, we compared here their impact on multiple cell types, evaluating i) histone acetylation and hydroxybutyrylation levels by immunoblotting, ii) transcriptional regulation of metabolic and inflammatory genes by quantitative PCR and iii) cytokine secretion profiles using proteome profiling array analysis. We confirm that butyrate is a strong HDAC inhibitor, a characteristic we could not identify in R-β-hydroxybutyrate in vivo nor in vitro. Butyrate had an extensive impact on gene transcription in rat myotubes, upregulating PGC1α, CPT1b, mitochondrial sirtuins (SIRT3-5), and the mitochondrial anti-oxidative genes SOD2 and catalase. In endothelial cells, butyrate suppressed gene expression and LPS-induced secretion of several pro-inflammatory genes, while R-β-hydroxybutyrate acted as a slightly pro-inflammatory molecule. Our observations indicate that butyrate induces transcriptional changes to a higher extent than R-β-hydroxybutyrate in rat myotubes and endothelial cells, in keep with its HDAC inhibitory activity. Also, in contrast with previous reports, R-β-hydroxybutyrate, while inducing histone β-hydroxybutyrylation, did not display a readily detectable HDAC inhibitor activity and exerted a slight pro-inflammatory action on endothelial cells.