microRNA‐379 couples glucocorticoid hormones to dysfunctional lipid homeostasis

In mammals, glucocorticoids (GCs) and their intracellular receptor, the glucocorticoid receptor (GR), represent critical checkpoints in the endocrine control of energy homeostasis. Indeed, aberrant GC action is linked to severe metabolic stress conditions as seen in Cushing's syndrome, GC therapy and certain components of the Metabolic Syndrome, including obesity and insulin resistance. Here, we identify the hepatic induction of the mammalian conserved microRNA (miR)‐379/410 genomic cluster as a key component of GC/GR‐driven metabolic dysfunction. Particularly, miR‐379 was up‐regulated in mouse models of hyperglucocorticoidemia and obesity as well as human liver in a GC/GR‐dependent manner. Hepatocyte‐specific silencing of miR‐379 substantially reduced circulating very‐low‐density lipoprotein (VLDL)‐associated triglyceride (TG) levels in healthy mice and normalized aberrant lipid profiles in metabolically challenged animals, mediated through miR‐379 effects on key receptors in hepatic TG re‐uptake. As hepatic miR‐379 levels were also correlated with GC and TG levels in human obese patients, the identification of a GC/GR‐controlled miRNA cluster not only defines a novel layer of hormone‐dependent metabolic control but also paves the way to alternative miRNA‐based therapeutic approaches in metabolic dysfunction.     

Increasing NAD Synthesis in Muscle via Nicotinamide Phosphoribosyltransferase Is Not Sufficient to Promote Oxidative Metabolism

The NAD biosynthetic precursors nicotinamide mononucleotide and nicotinamide riboside are reported to confer resistance to metabolic defects induced by high fat feeding in part by promoting oxidative metabolism in skeletal muscle. Similar effects are obtained by germ line deletion of major NAD-consuming enzymes, suggesting that the bioavailability of NAD is limiting for maximal oxidative capacity. However, because of their systemic nature, the degree to which these interventions exert cell- or tissue-autonomous effects is unclear. Here, we report a tissue-specific approach to increase NAD biosynthesis only in muscle by overexpressing nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in the salvage pathway that converts nicotinamide to NAD (mNAMPT mice). These mice display a ∼50% increase in skeletal muscle NAD levels, comparable with the effects of dietary NAD precursors, exercise regimens, or loss of poly(ADP-ribose) polymerases yet surprisingly do not exhibit changes in muscle mitochondrial biogenesis or mitochondrial function and are equally susceptible to the metabolic consequences of high fat feeding. We further report that chronic elevation of muscle NAD in vivo does not perturb the NAD/NADH redox ratio. These studies reveal for the first time the metabolic effects of tissue-specific increases in NAD synthesis and suggest that critical sites of action for supplemental NAD precursors reside outside of the heart and skeletal muscle.     

Two-step Ligand Binding in a (βα)8 Barrel Enzyme: SUBSTRATE-BOUND STRUCTURES SHED NEW LIGHT ON THE CATALYTIC CYCLE OF HisA*

HisA is a (βα)₈ barrel enzyme that catalyzes the Amadori rearrangement of N′-[(5′-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) to N′-((5′-phosphoribulosyl) formimino)-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) in the histidine biosynthesis pathway, and it is a paradigm for the study of enzyme evolution. Still, its exact catalytic mechanism has remained unclear. Here, we present crystal structures of wild type Salmonella enterica HisA (SeHisA) in its apo-state and of mutants D7N and D7N/D176A in complex with two different conformations of the labile substrate ProFAR, which was structurally visualized for the first time. Site-directed mutagenesis and kinetics demonstrated that Asp-7 acts as the catalytic base, and Asp-176 acts as the catalytic acid. The SeHisA structures with ProFAR display two different states of the long loops on the catalytic face of the structure and demonstrate that initial binding of ProFAR to the active site is independent of loop interactions. When the long loops enclose the substrate, ProFAR adopts an extended conformation where its non-reacting half is in a product-like conformation. This change is associated with shifts in a hydrogen bond network including His-47, Asp-129, Thr-171, and Ser-202, all shown to be functionally important. The closed conformation structure is highly similar to the bifunctional HisA homologue PriA in complex with PRFAR, thus proving that structure and mechanism are conserved between HisA and PriA. This study clarifies the mechanistic cycle of HisA and provides a striking example of how an enzyme and its substrate can undergo coordinated conformational changes before catalysis.     

Parvulin 17-catalyzed Tubulin Polymerization Is Regulated by Calmodulin in a Calcium-dependent Manner

Recently we have shown that the peptidyl-prolyl cis/trans isomerase parvulin 17 (Par17) interacts with tubulin in a GTP-dependent manner, thereby promoting the formation of microtubules. Microtubule assembly is regulated by Ca²⁺-loaded calmodulin (Ca²⁺/CaM) both in the intact cell and under in vitro conditions via direct interaction with microtubule-associated proteins. Here we provide the first evidence that Ca²⁺/CaM interacts also with Par17 in a physiologically relevant way, thus preventing Par17-promoted microtubule assembly. In contrast, parvulin 14 (Par14), which lacks only the first 25 N-terminal residues of the Par17 sequence, does not interact with Ca²⁺/CaM, indicating that this interaction is exclusive for Par17. Pulldown experiments and chemical shift perturbation analysis with ¹⁵N-labeled Par17 furthermore confirmed that calmodulin (CaM) interacts in a Ca²⁺-dependent manner with the Par17 N terminus. The reverse experiment with ¹⁵N-labeled Ca²⁺/CaM demonstrated that the N-terminal Par17 segment binds to both CaM lobes simultaneously, indicating that Ca²⁺/CaM undergoes a conformational change to form a binding channel between its two lobes, apparently similar to the structure of the CaM-smMLCK^796–815 complex. In vitro tubulin polymerization assays furthermore showed that Ca²⁺/CaM completely suppresses Par17-promoted microtubule assembly. The results imply that Ca²⁺/CaM binding to the N-terminal segment of Par17 causes steric hindrance of the Par17 active site, thus interfering with the Par17/tubulin interaction. This Ca²⁺/CaM-mediated control of Par17-assisted microtubule assembly may provide a mechanism that couples Ca²⁺ signaling with microtubule function.     

Science in Risk Assessment and Policy (SciRAP): An Online Resource for Evaluating and Reporting In Vivo (Eco)Toxicity Studies

(Eco)toxicity studies conducted according to internationally standardized test guidelines are often considered reliable by default and preferred as key evidence in regulatory risk assessment. At the same time regulatory agencies emphasize the use of all relevant (eco)toxicity data in the risk assessment process, including non-standard studies. However, there is a need to facilitate the use of such studies in regulatory risk assessment. Therefore, we propose a framework that facilitates a systematic and transparent evaluation of the reliability and relevance of (eco)toxicity in vivo studies for health and environmental risk assessment. The framework includes specific criteria to guide study evaluation, as well as a color-coding tool developed to aid the application of these criteria. In addition we provide guidance intended for researchers on how to report non-standard studies to ensure that they meet regulatory requirements. The intention of the evaluating and reporting criteria is to increase the usability of all relevant data that may fill information gaps in chemical risk assessments. The framework is publically available online, free of charge, at the Science in Risk Assessment and Policy (SciRAP) website: www.scirap.org. The aim of this article is to present the framework and resources available at the SciRAP website.     

Discovery of N-(1-adamantyl)-2-(4-alkylpiperazin-1-yl)acetamide derivatives as T-type calcium channel (Cav3.2) inhibitors

Chemical evolution of a HTS-based fragment hit resulted in the identification of N-(1-adamantyl)-2-[4-(2-tetrahydropyran-4-ylethyl)piperazin-1-yl]acetamide, a novel, selective T-type calcium channel (Ca(v)3.2) inhibitor with in vivo antihypertensive effect in rats.     

Pericytes: developmental, physiological, and pathological perspectives, problems, and promises

Pericytes, the mural cells of blood microvessels, have recently come into focus as regulators of vascular morphogenesis and function during development, cardiovascular homeostasis, and disease. Pericytes are implicated in the development of diabetic retinopathy and tissue fibrosis, and they are potential stromal targets for cancer therapy. Some pericytes are probably mesenchymal stem or progenitor cells, which give rise to adipocytes, cartilage, bone, and muscle. However, there is still confusion about the identity, ontogeny, and progeny of pericytes. Here, we review the history of these investigations, indicate emerging concepts, and point out problems and promise in the field of pericyte biology.     

Metabolic phenotyping of the Crohn's disease-like IBD etiopathology in the TNF(ΔARE/WT) mouse model

The underlying biochemical consequences of inflammatory bowel disease (IBD) on the systemic and gastrointestinal metabolism have not yet been fully elucidated but could help to better understand the disease pathogenesis and to identify tissue-specific markers associated with the different disease stages. Here, we applied a metabonomic approach to monitor metabolic events associated with the gradual development of Crohn's disease (CD)-like ileitis in the TNF(ΔARE/WT) mouse model. Metabolic profiles of different intestinal compartments from the age of 4 up to 24 weeks were generated by combining proton nuclear magnetic resonance ((1)H NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS). From 8 weeks onward, mice developed CD similar to the immune and tissue-related phenotype of human CD with ileal involvement, including ileal histological abnormalities, reduced fat mass and body weight, as well as hallmarks of malabsorption with higher energy wasting. The metabonomic approach highlighted shifts in the intestinal lipid metabolism concomitant to the histological onset of inflammation. Moreover, the advanced disease status was characterized by a significantly altered metabolism of cholesterol, triglycerides, phospholipids, plasmalogens, and sphingomyelins in the inflamed tissue (ileum) and the adjacent intestinal parts (proximal colon). These results describe different biological processes associated with the disease onset, including modifications of the general cell membrane composition, alteration of energy homeostasis, and finally the generation of inflammatory lipid mediators. Taken together, this provides novel insights into IBD-related alterations of specific lipid-dependant processes during inflammatory states.