Deletion of fatty acid transport protein 2 (FATP2) in the mouse liver changes the metabolic landscape by increasing the expression of PPARα-regulated genes.

Fatty acid transport protein 2 (FATP2) is highly expressed in the liver, small intestine, and kidney, where it functions in both the transport of exogenous long-chain fatty acids and the activation of very-long-chain fatty acids. Here, using a murine model, we investigated the phenotypic impacts of deleting FATP2, followed by a transcriptomic analysis using unbiased RNA-Seq to identify concomitant changes in the liver transcriptome. WT and FATP2-null (Fatp2-/-) mice (5 weeks) were maintained on a standard chow diet for 6 weeks. The Fatp2-/- mice had reduced weight gain, lowered serum triglyceride, and increased serum cholesterol levels and attenuated dietary fatty acid absorption. Transcriptomic analysis of the liver revealed 258 differentially expressed genes in male Fatp2-/- mice and a total of 91 in female Fatp2-/- mice. These genes mapped to the following gene ontology categories: fatty acid degradation, peroxisome biogenesis, fatty acid synthesis, and retinol and arachidonic acid metabolism. Targeted RT-quantitative PCR verified the altered expression of selected genes. Of note, most of the genes with increased expression were known to be regulated by peroxisome proliferator-activated receptor α (PPARα), suggesting that FATP2 activity is linked to a PPARα-specific proximal ligand. Targeted metabolomic experiments in the Fatp2-/- liver revealed increases of total C16:0, C16:1, and C18:1 fatty acids; increases in lipoxin A4 and prostaglandin J2; and a decrease in 20-hydroxyeicosatetraenoic acid. We conclude that the expression of FATP2 in the liver broadly affects the metabolic landscape through PPARα, indicating that FATP2 provides an important role in liver lipid metabolism through its transport or activation activities.

Anti-TNFα treatment in Crohn's disease: Impact on hepatic steatosis, gut-derived hormones and metabolic status.

BACKGROUND AND AIMS: An association between Crohn's disease (CD) and hepatic steatosis has been reported. However, the underlying mechanisms of steatosis progression in CD are not clear. Among the most effective CD treatments are agents that inhibit Tumor-Necrosis-Factor (TNF) activity, yet it is unclear why anti-TNFα agents would affect steatosis in CD. Recent studies suggest that microbiome can affect both, CD and steatosis pathogenesis. Therefore, we here analysed a potential relationship between anti-TNF treatment and hepatic steatosis in CD, focusing on the gut-liver axis. METHODS: This cross-sectional study evaluated patients with established CD, with and without anti-TNFα treatment, analysing serum markers of liver injury, measurement of transient elastography, controlled attenuation parameter (CAP) and MRI for fat detection. Changes in lipid and metabolic profiles were assessed by serum and stool lipidomics and metabolimics. Additionally, we analysed gut microbiota composition and mediators of bile acid (BA) signalling via stool and serum analysis. RESULTS: Patients on anti-TNFα treatment had less hepatic steatosis as assessed by CAP and MRI. Serum FGF19 levels were significantly higher in patients on anti-TNFα therapy and associate with reduced steatosis and increased bowel motility. Neutral lipids including triglycerides were reduced in the serum of patients on anti-TNF treatment. Bacteria involved in BA metabolism and FGF19 regulation, including Firmicutes, showed group-specific alterations with low levels in patients without anti-TNFα treatment. Low abundance of Firmicutes was associated with higher triglyceride levels. CONCLUSIONS: Anti-TNFα treatment is associated with reduced steatosis, lower triglyceride levels, alterations in FXR-signalling (eg FGF19) and microbiota composition in CD.

Remodeling of Chlamydomonas Metabolism Using Synthetic Inducers Results in Lipid Storage during Growth.

Microalgae accumulate lipids during stress such as that of nutrient deprivation, concomitant with cessation of growth and depletion of chloroplasts. By contrast, certain small chemical compounds selected by high-throughput screening in Chlamydomonas reinhardtii can induce lipid accumulation during growth, maintaining biomass. Comprehensive pathway analyses using proteomics, transcriptomics, and metabolomics data were acquired from Chlamydomonas cells grown in the presence of one of two structurally distinct lipid activators. WD10784 stimulates both starch and lipid accumulation, whereas WD30030-treated cells accumulate only lipids. The differences in starch accumulation are largely due to differential effects of the two compounds on substrate levels that feed into starch synthesis and on genes encoding starch metabolic enzymes. The compounds had differential effects on photosynthesis, respiration, and oxidative stress pathways. Cells treated with WD10784 showed slowed growth over time and reduced abundance of photosynthetic proteins, decreased respiration, and increased oxidative stress proteins, glutathione, and reactive oxygen species specific to this compound. Both compounds maintained central carbon and nitrogen metabolism, including the tricarboxylic acid cycle, glycolysis, respiration, and the Calvin-Benson-Bassham cycle. There were few changes in proteins and transcripts related to fatty acid biosynthesis, whereas proteins and transcripts for triglyceride production were elevated, suggesting that lipid synthesis is largely driven by substrate availability. This study reports that the compound WD30030 and, to a lesser extent WD10784, increases lipid and lipid droplet synthesis and storage without restricting growth or biomass accumulation by mechanisms that are substantially different from nutrient deprivation.

Combining Mass Spectrometry and NMR Improves Metabolite Detection and Annotation.

Despite inherent complementarity, nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) are routinely separately employed to characterize metabolomics samples. More troubling is the erroneous view that metabolomics is better served by exclusively utilizing MS. Instead, we demonstrate the importance of combining NMR and MS for metabolomics by using small chemical compound treatments of Chlamydomonas reinhardtii as an illustrative example. A total of 102 metabolites were detected (82 by gas chromatography-MS, 20 by NMR, and 22 by both techniques). Out of these, 47 metabolites of interest were identified: 14 metabolites were uniquely identified by NMR, and 16 metabolites were uniquely identified by GC-MS. A total of 17 metabolites were identified by both NMR and GC-MS. In general, metabolites identified by both techniques exhibited similar changes upon compound treatment. In effect, NMR identified key metabolites that were missed by MS and enhanced the overall coverage of the oxidative pentose phosphate pathway, Calvin cycle, tricarboxylic acid cycle, and amino acid biosynthetic pathways that informed on pathway activity in central carbon metabolism, leading to fatty-acid and complex-lipid synthesis. Our study emphasizes a prime advantage of combining multiple analytical techniques: the improved detection and annotation of metabolites.

Identification and Metabolite Profiling of Chemical Activators of Lipid Accumulation in Green Algae.

Microalgae are proposed as feedstock organisms useful for producing biofuels and coproducts. However, several limitations must be overcome before algae-based production is economically feasible. Among these is the ability to induce lipid accumulation and storage without affecting biomass yield. To overcome this barrier, a chemical genetics approach was employed in which 43,783 compounds were screened against Chlamydomonas reinhardtii, and 243 compounds were identified that increase triacylglyceride (TAG) accumulation without terminating growth. Identified compounds were classified by structural similarity, and 15 were selected for secondary analyses addressing impacts on growth fitness, photosynthetic pigments, and total cellular protein and starch concentrations. TAG accumulation was verified using gas chromatography-mass spectrometry quantification of total fatty acids, and targeted TAG and galactolipid measurements were performed using liquid chromatography-multiple reaction monitoring/mass spectrometry. These results demonstrated that TAG accumulation does not necessarily proceed at the expense of galactolipid. Untargeted metabolite profiling provided important insights into pathway shifts due to five different compound treatments and verified the anabolic state of the cells with regard to the oxidative pentose phosphate pathway, Calvin cycle, tricarboxylic acid cycle, and amino acid biosynthetic pathways. Metabolite patterns were distinct from nitrogen starvation and other abiotic stresses commonly used to induce oil accumulation in algae. The efficacy of these compounds also was demonstrated in three other algal species. These lipid-inducing compounds offer a valuable set of tools for delving into the biochemical mechanisms of lipid accumulation in algae and a direct means to improve algal oil content independent of the severe growth limitations associated with nutrient deprivation.

Integrated quantitative analysis of nitrogen stress response in Chlamydomonas reinhardtii using metabolite and protein profiling.

Nitrogen starvation induces a global stress response in microalgae that results in the accumulation of lipids as a potential source of biofuel. Using GC-MS-based metabolite and iTRAQ-labeled protein profiling, we examined and correlated the metabolic and proteomic response of Chlamydomonas reinhardtii under nitrogen stress. Key amino acids and metabolites involved in nitrogen sparing pathways, methyl group transfer reactions, and energy production were decreased in abundance, whereas certain fatty acids, citric acid, methionine, citramalic acid, triethanolamine, nicotianamine, trehalose, and sorbitol were increased in abundance. Proteins involved in nitrogen assimilation, amino acid metabolism, oxidative phosphorylation, glycolysis, TCA cycle, starch, and lipid metabolism were elevated compared with nonstressed cultures. In contrast, the enzymes of the glyoxylate cycle, one carbon metabolism, pentose phosphate pathway, the Calvin cycle, photosynthetic and light harvesting complex, and ribosomes were reduced. A noteworthy observation was that citrate accumulated during nitrogen stress coordinate with alterations in the enzymes that produce or utilize this metabolite, demonstrating the value of comparing protein and metabolite profiles to understand complex patterns of metabolic flow. Thus, the current study provides unique insight into the global metabolic adjustments leading to lipid storage during N starvation for application toward advanced biofuel production technologies.

Phagocytosis in the retina promotes local insulin production in the eye.

The retina is highly metabolically active, relying on glucose uptake and aerobic glycolysis. Situated in close contact to photoreceptors, a key function of cells in the retinal pigment epithelium (RPE) is phagocytosis of damaged photoreceptor outer segments (POS). Here we identify RPE as a local source of insulin in the eye that is stimulated by POS phagocytosis. We show that Ins2 messenger RNA and insulin protein are produced by RPE cells and that this production correlates with RPE phagocytosis of POS. Genetic deletion of phagocytic receptors ('loss of function') reduces Ins2, whereas increasing the levels of the phagocytic receptor MerTK ('gain of function') increases Ins2 production in male mice. Contrary to pancreas-derived systemic insulin, RPE-derived local insulin is stimulated during starvation, which also increases RPE phagocytosis. Global or RPE-specific Ins2 gene deletion decreases retinal glucose uptake in starved male mice, dysregulates retinal physiology, causes defects in phototransduction and exacerbates photoreceptor loss in a mouse model of retinitis pigmentosa. Collectively, these data identify RPE cells as a phagocytosis-induced local source of insulin in the retina, with the potential to influence retinal physiology and disease.

High-Throughput Profiling of Metabolic Phenotypes Using High-Resolution GC-MS.

Metabolite profiling provides insights into the metabolic signatures, which themselves are considered as phonotypes closely related to the agronomic and phenotypic traits such as yield, nutritional values, stress resistance, and nutrient use efficiency. GC-MS is a sensitive and high-throughput analytical platform and has been proved to be a vital tool for the analysis of primary metabolism to provide an overview of cellular and organismal metabolic status. The potential of GC-MS metabolite profiling as a tool for detecting metabolic changes in plants grown in a high-throughput plant phenotyping platform was explored. In this chapter, we describe an integrated workflow of semi-targeted GC-high-resolution (HR)-time-of-flight (TOF)-MS metabolomics with both the analytical and computational steps, focusing mainly on the sample preparation, GC-HR-TOF-MS analysis part, and data analysis for plant phenotyping efforts.

Longitudinal Metabolomics and Lipidomics Analyses Reveal Alterations Associated with Envenoming by Bothrops asper and Daboia russelii in an Experimental Murine Model.

Longitudinal metabolomics and lipidomics analyses were carried out on the blood plasma of mice injected intramuscularly with venoms of the viperid species Bothrops asper or Daboia russelii. Blood samples were collected 1, 3, 6, and 24 h after venom injection, and a control group of non-envenomed mice was included. Significant perturbations in metabolomics and lipidomics were observed at 1, 3, and 6 h, while values returned close to those of control mice by 24 h, hence reflecting a transient pattern of metabolic disturbance. Both venoms induced significant changes in amino acids, as well as in several purines and pyrimidines, and in some metabolites of the tricarboxylic acid cycle. KEGG analysis of metabolic pathways that showed those with the greatest change included aminoacyl tRNA synthesis and amino acid biosynthesis and metabolism pathways. With regard to lipid metabolism, there was an increase in triglycerides and some acyl carnitines and a concomitant drop in the levels of some phospholipids. In addition, envenomed mice had higher levels of cortisol, heme, and some oxidative stress markers. The overall pattern of metabolic changes in envenomed mice bears similarities with the patterns described in several traumatic injuries, thus underscoring a metabolic response/adaptation to the injurious action of the venoms.

Genome of Paspalum vaginatum and the role of trehalose mediated autophagy in increasing maize biomass.

A number of crop wild relatives can tolerate extreme stress to a degree outside the range observed in their domesticated relatives. However, it is unclear whether or how the molecular mechanisms employed by these species can be translated to domesticated crops. Paspalum (Paspalum vaginatum) is a self-incompatible and multiply stress-tolerant wild relative of maize and sorghum. Here, we describe the sequencing and pseudomolecule level assembly of a vegetatively propagated accession of P. vaginatum. Phylogenetic analysis based on 6,151 single-copy syntenic orthologues conserved in 6 related grass species places paspalum as an outgroup of the maize-sorghum clade. In parallel metabolic experiments, paspalum, but neither maize nor sorghum, exhibits a significant increase in trehalose when grown under nutrient-deficit conditions. Inducing trehalose accumulation in maize, imitating the metabolic phenotype of paspalum, results in autophagy dependent increases in biomass accumulation.

Untargeted Metabolomics Reveals Species-Specific Metabolite Production and Shared Nutrient Consumption by Pseudomonas aeruginosa and Staphylococcus aureus.

While bacterial metabolism is known to impact antibiotic efficacy and virulence, the metabolic capacities of individual microbes in cystic fibrosis lung infections are difficult to disentangle from sputum samples. Here, we show that untargeted metabolomic profiling of supernatants of multiple strains of Pseudomonas aeruginosa and Staphylococcus aureus grown in monoculture in synthetic cystic fibrosis media (SCFM) reveals distinct species-specific metabolic signatures despite intraspecies metabolic variability. We identify a set of 15 metabolites that were significantly consumed by both P. aeruginosa and S. aureus, suggesting that nutrient competition has the potential to impact community dynamics even in the absence of other pathogen-pathogen interactions. Finally, metabolites that were uniquely produced by one species or the other were identified. Specifically, the virulence factor precursor anthranilic acid, as well as the quinoline 2,4-quinolinediol (DHQ), were robustly produced across all tested strains of P. aeruginosa. Through the direct comparison of the extracellular metabolism of P. aeruginosa and S. aureus in a physiologically relevant environment, this work provides insight toward the potential for metabolic interactions in vivo and supports the development of species-specific diagnostic markers of infection. IMPORTANCE Interactions between P. aeruginosa and S. aureus can impact pathogenicity and antimicrobial efficacy. In this study, we aim to better understand the potential for metabolic interactions between P. aeruginosa and S. aureus in an environment resembling the cystic fibrosis lung. We find that S. aureus and P. aeruginosa consume many of the same nutrients, suggesting that metabolic competition may play an important role in community dynamics during coinfection. We further identify metabolites uniquely produced by either organism with the potential to be developed into species-specific biomarkers of infection in the cystic fibrosis lung.

Transcriptome analysis-identified long noncoding RNA CRNDE in maintaining endothelial cell proliferation, migration, and tube formation.

Obesity is a leading risk factor for type-2 diabetes. Diabetes often leads to the dysregulation of angiogenesis, although the mechanism is not fully understood. Previously, long noncoding RNAs (lncRNAs) have been found to modulate angiogenesis. In this study, we asked how the expression levels of lncRNAs change in endothelial cells in response to excessive palmitic acid treatment, an obesity-like condition. Bioinformatics analysis revealed that 305 protein-coding transcripts were upregulated and 70 were downregulated, while 64 lncRNAs were upregulated and 46 were downregulated. Gene ontology and pathway analysis identified endoplasmic reticulum stress, HIF-1 signaling, and Toll-like receptor signaling as enriched after palmitic acid treatment. Moreover, we newly report enrichment of AGE-RAGE signaling pathway in diabetic complications, IL-17 signaling, and cysteine and methionine metabolism by palmitic acid. One lncRNA, Colorectal Neoplasia Differentially Expressed (CRNDE), was selected for further investigation. Palmitic acid induces CRNDE expression by 1.9-fold. We observed that CRNDE knockdown decreases endothelial cell proliferation, migration, and capillary tube formation. These decreases are synergistic under palmitic acid stress. These data demonstrated that lncRNA CRNDE is a regulator of endothelial cell proliferation, migration, and tube formation in response to palmitic acid, and a potential target for therapies treating the complications of obesity-induced diabetes.

Innovations in improving lipid production: Algal chemical genetics.

Perturbation of a biological system with small molecules to achieve a desired phenotype or activity is commonly referred as chemical genetics. In pharmaceutical discovery, this approach is most often employed in target-based screening but in plants systems the focus is primarily on phenotypic selection for commercially relevant phenotype generation such as crop improvement or disease and pathogen resistance. Likewise, algae are considered feedstock organisms for viable and sustainable biofuels and other high value products with commercial applications. Algal triacylglycerol synthesis is therefore an important target for chemical genetics using high throughput technologies. In this review, efforts are directed towards summarizing our present understanding of the regulation of algal triacylglycerol biosynthesis, highlighting critical enzymes in lipid and carbon metabolism that may be manipulated to increase lipid metabolism in algae. These enzymes and pathways are targets for chemical genetics with the focus on selection of small molecules as tools to improve triacylglycerol storage. Using case studies, we summarize how chemical genetics is being used in plant and microalgal systems to address these critical problems.

Quantitative analysis of UV-A shock and short term stress using iTRAQ, pseudo selective reaction monitoring (pSRM) and GC-MS based metabolite analysis of the cyanobacterium Nostoc punctiforme ATCC 29133.

A quantitative proteomics and metabolomics analysis was performed using iTRAQ, HPLC and GC-MS in the filamentous cyanobacterium Nostoc punctiforme ATCC 29133 to understand the effect of short and long term UV-A exposure. Changes in the proteome were measured for short-term stress (4-24h) using iTRAQ. Changes in the photosynthetic pigments and intracellular metabolites were observed at exposures of up to 7days (pigments) and up to 11days (intracellular metabolites). To assess iTRAQ measurement quality, pseudo selected reaction monitoring (pSRM) was used, with this confirming underestimation of protein abundance levels by iTRAQ. Our results suggest that short term UV-A radiation lowers the abundance of PS-I and PS-II proteins. We also observed an increase in abundance of intracellular redox homeostasis proteins and plastocyanin. Additionally, we observed statistically significant changes in scytonemin, Chlorophyll A, astaxanthin, zeaxanthin, and ß-carotene. Assessment of intracellular metabolites showed significant changes in several, suggesting their potential role in the Nostoc's stress mitigation strategy. Cyanobacteria under UV-A radiation have reduced growth due to intensive damage to essential functions, but the organism shows a defense response by remodeling bioenergetics pathway, induction of the UV protection compound scytonemin and increased levels of proline and tyrosine as a mitigation response. BIOLOGICAL SIGNIFICANCE: The effect of UV-A radiation on the proteome and intracellular metabolites of N. punctiforme ATCC 29133 including photosynthetic pigments has been described. We also verify the expression of 13 iTRAQ quantified protein using LC-pSRM. Overall we observed that UV-A radiation has a drastic effect on the photosynthetic machinery, photosynthetic pigments and intracellular amino acids. As a mitigation strategy against UV-A radiation, proline, glycine, and tyrosine were accumulated.

Systems biology of cyanobacterial secondary metabolite production and its role in drug discovery.

BACKGROUND: Cyanobacteria continue to be an important source of compounds that show unprecedented biological activities of pharmaceutical interest. Cyanobacterial metabolites show an interesting and exciting range of biological activities ranging from antimicrobial and immunosuppressant to anticancer and anti-HIV. OBJECTIVE: This review explores the possibilities of applying systems biology approaches for harnessing these compounds as drug leads, primarily produced through large multimodular non-ribosomal peptide synthetase (NRPS), polyketide synthase (PKS) and mixed NRPS-PKS enzymatic systems. METHODS: A brief survey of the strategies for in silico analysis for drug target identification using genomic and high-throughput proteomics data, virtual screening and receptor-ligand docking based approaches are also discussed. CONCLUSION: We conclude with an outlook on how the field will evolve, especially in partnership with the new engineering-based, more endpoint exploitative paradigm of synthetic biology.