Down-regulation Of Metabolism Research Articles

Effects of IC50 dose of retinol on metabolomics of RAW264.7 cells.

Vitamin A is one of the most multifunctional vitamins in normal human physiology and is involved in several basic physiological processes from embryonic development to adulthood, such as embryogenesis, vision, immunity, cell differentiation, and proliferation. In this study, we conducted 1 H- NMR to evaluate the metabolomic changes in RAW264.7 cells after treatment with retinol at an IC50 dose to identify its effects on the differential metabolites and main metabolic pathways. Our results showed that the IC50 dose (140μM) of retinol affected the metabolism of RAW264.7 cells, with a total of 22 differential metabolites identified via 1 H-NMR, including amino acids, sugars, organic acids, glutathione, glycerin, and creatine. Additionally, multiple metabolic pathways were affected by retinol treatment, including downregulation of amino acid biosynthesis, protein synthesis, and pyruvate metabolism. We speculate that the cytotoxicity of retinol at the IC50 dose is attributed to mitochondrial dysfunction as a result of oxidative stress or lipid peroxidation. PRACTICAL APPLICATIONS: With the general improvement of people's living standards, people use dietary supplements to improve the level of retinol to prevent non-specific diseases. But there are more and more cases of acute or chronic poisoning caused by excessive intake of vitamin A. Therefore, it is necessary to study the toxicity of vitamin A, and more attentionshould be paidto the excessive intake of vitamin A. From the perspective of metabolomics, this experiment studies the adverse effects of high dose retinol through the changes of metabolites and metabolic pathwaysat the cellular level.This study will assist further analyses of the toxic mechanism of excessive retinol as fortified foods and nutrient supplementation.

469-P: ROCK1/AMPK Axis Regulates the Development of Diabetic Kidney Disease via Modulation of Fatty Acid Metabolism

The small GTPase Rho and its effector Rho-kinase are involved in the pathogenesis of diabetic kidney disease. Accumulating evidence shows that renal dysfunction in diabetic patient is associated with abnormal fatty acid oxidation in the kidney. However, the interaction of Rho-kinase and fatty acid oxidation in diabetic kidney remain unclear. Glomeruli isolated from type 2 diabetic db/db mice demonstrated decreased gene expression of fatty acid oxidation mediators such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), CD36, carnitine palmitoyltransferase 1A (CPT1A). Chemical inhibition of Rho-kinase restored expression of fatty acid oxidation-related genes in both isolated glomeruli and cultured mesangial cells. An investigation of mechanisms underlying this observation revealed activated Rho-kinase functions through the phosphorylation of AMPK and increased expression of PGC-1α. Extracellular flux analyzer demonstrated that Rho-kinase inhibition improves TGF-β-induced mitochondrial dysfunction. Furthermore, fluorescence histochemistry showed that Rho-kinase inhibitors suppresses ROS production as a result of mitochondrial damage due to abnormal fatty acid metabolism. Knockdown by small interfering RNA against two Rho-kinase isoforms, ROCK1 and ROCK2, showed that ROCK1 but not ROCK2 controls this metabolic machinery. Consistent with this result, mesangial cells isolated from ROCK1 deficient mice were protected from TGF-β-mediated downregulation of fatty acid metabolism. These observations indicate that ROCK1 is a key player in the development of diabetic renal injury. Glomerular ROCK1 may be a potential therapeutic target for the treatment of diabetic kidney disease. Disclosure Y. Nagai: None. K. Matoba: Research Support; Self; Sanofi K.K., Takeda Pharmaceutical Company Limited. Y. Takeda: None. T. Akamine: None. D. Kawanami: None. K. Utsunomiya: None. R. Nishimura: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc. Speaker’s Bureau; Self; Abbott, AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly Japan K.K., Medtronic, Merck & Co., Inc., Novo Nordisk Inc., Sanofi K.K., Takeda Pharmaceutical Company Limited.

Indole-3-carbinol alleviates carbon tetrachloride-induced liver injury by inhibiting inflammatory response and regulating lipid metabolism

The hepatoprotective effects of indole-3-carbinol (I3C), one of the byproducts of the breakdown of glucosinolate glucobrassicin, were examined in mice with carbon tetrachloride (CCl4)-induced hepatotoxicity. I3C was administered orally at a dose of 50 or 100 mg/kg to C57BL/6 mice for 3 consecutive days with or without a subsequent single oral administration of CCl4 (1.5 mL/kg). I3C treatment significantly suppressed the increases in alanine aminotransferase and aspartate aminotransferase levels in the sera of CCl4-injured mice. In addition, I3C treatment markedly suppressed CCl4-induced hepatic inflammation, by suppressing the activation of macrophages and Kupffer cells in the liver. To examine the effects of I3C on lipid accumulation, 3T3-L1 pre-adipocytes were differentiated with I3C or vehicle control. I3C treatment reduced lipid accumulation, which appeared to be associated with the downregulation of adipogenic transcription factors, such as CCAAT/enhancer-binding protein α, peroxisome proliferator-activated receptor γ, and lipid accumulation-related genes, including adipocyte protein-2. Taken together, the present study suggest that I3C treatment reduces hepatocyte death and lipid accumulation in hepatocytes in CCl4-induced hepatic injury with concurrent suppression of macrophages/Kupffer cells, which was further evidenced by I3C-treated pre-adipocytes in vitro, through downregulation of lipid metabolism.

Au nanorod-induced NLRP3 inflammasome activation is mediated by ER stress

Abstract The widespread and increasing use of engineered nanomaterials (ENM) increases the risk of human exposure, generating concern that ENM may provoke adverse health effects. In this respect, their physicochemical characteristics are critical. The immune system may respond to specific ENM properties by inflammatory reactions. Inflammasome activation has drawn significant attention since inflammasomes, especially NLRP3 respond to a wide range of stimuli including nanoparticles, and their activation is associated with various inflammatory diseases, including lung fibrosis, obesity and type-2 diabetes. Endoplasmic reticulum (ER) stress has been reported as one of the mechanisms underlying NLRP3 inflammasome activation. In this study, PEGylated Au ENM of ≈60 nm, having different shapes (stars, spheres and rods) were extensively characterized, and tested for possible LPS contamination. PMA-activated THP-1 cells were exposed to these ENM, and cell viability and IL-1β production were measured to assess NLRP3 activation. In addition, the exposed cells were subjected to gene expression analysis using microarray analysis to investigate related signalling pathway regulation. PEGylated Au nanorods (NR), but not nanostars or nanospheres, showed NLRP3 inflammasome activation. Cells deficient in the NLRP3 scaffold or ASC adaptor did not show this effect. Only NR-exposed cells showed down-regulation of ER-associated cholesterol metabolism. This finding may suggest that ER stress mediates NLRP3 inflammasome activation by Au NR. Supported by the EU funded project FutureNanoNeeds (Grant agreement N° 604602).

Down-Regulation of Mitochondrial Metabolism after Tendon Release Primes Lipid Accumulation in Rotator Cuff Muscle

Atrophy and fat accumulation are debilitating aspects of muscle diseases and are rarely prevented. Using a vertical approach combining anatomic techniques with omics methodology in a tenotomy-induced sheep model of rotator cuff disease, we tested whether mitochondrial dysfunction is implicated in muscle wasting and perturbed lipid metabolism, speculating that both can be prevented by the stimulation of β-oxidation with l-carnitine. The infraspinatus muscle lost 22% of its volume over the first 6 weeks after tenotomy before the area-percentage of lipid increased from 8% to 18% at week 16. Atrophy was associated with the down-regulation of mitochondrial transcripts and protein and a slow-to-fast shift in muscle composition. Correspondingly, amino acid levels were increased 2 weeks after tendon release, when the levels of high-energy phosphates and glycerophospholipids were lowered. l-Carnitine administration (0.9 g/kg per day) prevented atrophy over the first 2 weeks, and mitigated alterations of glutamate, glycerophospholipids, and carnitine levels in released muscle, but did not prevent the level decrease in high-energy phosphates or protein constituents of mitochondrial respiration, promoting the accumulation of longer lipids with an increasing saturation. We conclude that the early phase of infraspinatus muscle degeneration after tendon release involves the elimination of oxidative characteristics associated with an aberrant accumulation of lipid species but is largely unrelated to the prevention of atrophy with oral l-carnitine administration.

High rumen degradable starch decreased goat milk fat via trans-10, cis-12 conjugated linoleic acid-mediated downregulation of lipogenesis genes, particularly, INSIG1

BackgroundStarch is an important substance that supplies energy to ruminants. To provide sufficient energy for high-yielding dairy ruminants, they are typically fed starch-enriched diets. However, starch-enriched diets have been proven to increase the risk of milk fat depression (MFD) in dairy cows. The starch present in ruminant diets could be divided into rumen-degradable starch (RDS) and rumen escaped starch (RES) according to their different degradation sites (rumen or intestine). Goats and cows have different sensitivities to MFD. Data regarding the potential roles of RDS in milk fat synthesis in the mammary tissue of dairy goats and in regulating the occurrence of MFD are limited.ResultsEighteen Guanzhong dairy goats (day in milk = 185 ± 12 d) with similar parity, weight, and milk yield were selected and randomly assigned to one of three groups (n = 6), which were fed an LRDS diet (Low RDS = 20.52%), MRDS diet (Medium RDS = 22.15%), or HRDS diet (High RDS = 24.88%) for 5 weeks. Compared with that of the LRDS group, the milk fat contents in the MRDS and HRDS groups significantly decreased. The yields of short-, medium- and long-chain fatty acids decreased in the HRDS group. Furthermore, increased RDS significantly decreased ruminal B. fibrisolvens and Pseudobutyrivibrio abundances and increased the trans-10, cis-12 conjugated linoleic acid (CLA) and trans-10 C18:1 contents in the rumen fluid.A multiomics study revealed that the HRDS diet affected mammary lipid metabolism down-regulation of ACSS2, MVD, AGPS, SCD5, FADS2, CERCAM, SC5D, HSD17B7, HSD17B12, ATM, TP53RK, GDF1 and LOC102177400. Remarkably, the significant decrease of INSIG1, whose expression was depressed by trans-10, cis-12 CLA, could reduce the activity of SREBP and, consequently, downregulate the downstream gene expression of SREBF1.ConclusionsHRDS-induced goat MFD resulted from the downregulation of genes involved in lipogenesis, particularly, INSIG1. Specifically, even though the total starch content and the concentrate-to-fiber ratio were the same as those of the high-RDS diet, the low and medium RDS diets did not cause MFD in lactating goats.

Using Electron Paramagnetic Resonance Spectroscopy to Determine the Role of Free‐Radical Protection in Tardigrade Survival Mechanisms

As aeronautic technology advances, so does the possibility of long‐term space travel and exploration. However, in order to make significant strides, it is first necessary to determine how to survive the antagonistic space environment, including temperature extremes, UVC radiation, and vacuum pressures, among others. Tardigrades are microscopic invertebrates that possess the ability to adapt to extreme environments through the formation of a tun, a dormant structure resulting from the downregulation of metabolism. This ability to survive under extreme conditions, including arctic temperatures, extreme heat, and a pure vacuum, makes tardigrades a useful model organism for space survival. By increasing our understanding of their survival mechanisms on a fundamental level, we may be able to generate technologies that protect humans in space from diseases caused by ROS and the damaging effects of oxidative stress.Previous research has examined the physiological response of tardigrades subjected to freezes and irradiation that model outer Earth conditions. In these studies, tardigrades were shown to enter tun formation through cryptobiosis, especially in response to physical stressors causing desiccation. Another study assessed tardigrades’ intergalactic survival capability by sending a culture into the vacuum of space, after which the cultures showed no loss of viability. We hypothesize that tardigrades have a specific, protective mechanism to respond to oxidative stress through the increased expression of superoxide dismutases (SODs) to break down potentially harmful ROS, which is supported by previous studies showing both the formation of superoxide radicals as well as an increase in SODs in desiccated animals.In this study, X‐band continuous wave electron paramagnetic resonance (EPR) spectroscopy was used to measure the presence of superoxide radicals in the tardigrade Hypsibius exemplaris before and after exposure to environmental stressors. Tardigrades were subjected to a combination of UVA and UVB irradiation before the addition of a spin probe, and the resulting EPR spectrum was compared to a nonirradiated H. exemplaris control. Results show that the tardigrades generated less ROS following environmental stress exposure, supporting the hypothesis of a protective mechanism to mediate radical production when subject to harsh conditions. Studies concerning other physical and chemical stressors, as well as examining radical production in tardigrades upon inducing tun formation, are ongoing. Further experiments also include determining specific activities of proteinaceous antioxidants like SOD before and after these exposures.Support or Funding InformationJ.R.C was supported the SURE Program funded through the West Virginia Research Challenge Fund, and administered by the West Virginia HEPC, DSR, Award Number DSR.17.09,the WV NASA Space Grant Consortium's NASA WV Space Grant Undergraduate Fellowship Program and from a Marshall University Creative Discover & Undergraduate Research Award.D.R.J.K. and A.L.S. were supported by the National Science Foundation under Cooperative Agreement No. OIA‐1458952.

Deoxycholic Acid-Induced Gut Dysbiosis Disrupts Bile Acid Enterohepatic Circulation and Promotes Intestinal Inflammation.

A Western diet is a risk factor for the development of inflammatory bowel disease (IBD). High levels of fecal deoxycholic acid (DCA) in response to a Western diet contribute to bowel inflammatory injury. However, the mechanism of DCA in the natural course of IBD development remains unanswered. The aim of this study is to investigate the effect of DCA on the induction of gut dysbiosis and its roles in the development of intestinal inflammation. Wild-type C57BL/6J mice were fed an AIN-93G diet, either supplemented with or without 0.2% DCA, and killed at 24weeks. Distal ileum and colon tissues were assessed by histopathological analysis. Hepatic and ileal gene expression was examined by qPCR, and the gut microbiota was analyzed by high-throughput 16S rRNA gene sequencing. HPLC-MS was used for fecal bile acid quantification. Mice fed the DCA-supplemented diet developed focal areas of ileal and colonic inflammation, accompanied by alteration of the composition of the intestinal microbiota and accumulation of fecal bile acids. DCA-induced dysbiosis decreased the deconjugation of bile acids, and this regulation was associated with the repressed expression of target genes in the enterohepatic farnesoid X receptor-fibroblast growth factor (FXR-FGF15) axis, leading to upregulation of hepatic de novo bile acid synthesis. These results suggest that DCA-induced gut dysbiosis may act as a key etiologic factor in intestinal inflammation, associated with bile acid metabolic disturbance and downregulation of the FXR-FGF15 axis.

Effect of aging and obesity on GLUT12 expression in small intestine, adipose tissue, muscle, and kidney and its regulation by docosahexaenoic acid and exercise in mice.

Obesity is characterized by excessive fat accumulation and inflammation. Aging has also been characterized as an inflammatory condition, frequently accompanied by accumulation of visceral fat. Beneficial effects of exercise and n-3 long-chain polyunsaturated fatty acids in metabolic disorders have been described. Glucose transporter 12 (GLUT12) is one of the less investigated members of the GLUT family. Glucose, insulin, and tumor necrosis factor alpha (TNF-α) induce GLUT12 translocation to the membrane in muscle, adipose tissue, and intestine. We aimed to investigate GLUT12 expression in obesity and aging, and under diet supplementation with docosahexaenoic acid (DHA) alone or in combination with physical exercise in mice. Aging increased GLUT12 expression in intestine, kidney, and adipose tissue, whereas obesity reduced it. No changes on the transporter occurred in skeletal muscle. In obese 18-month-old mice, DHA further decreased GLUT12 in the 4 organs. Aerobic exercise alone did not modify GLUT12, but the changes triggered by exercise were able to prevent the DHA-diminishing effect, and almost restored GLUT12 basal levels. In conclusion, the downregulation of metabolism in aging would be a stimulus to upregulate GLUT12 expression. Contrary, obesity, an excessive energy condition, would induce GLUT12 downregulation. The combination of exercise and DHA would contribute to restore basal function of GLUT12. Novelty In small intestine, kidney and adipose tissue aging increases GLUT12 protein expression whereas obesity reduces it. Dietary DHA decreases GLUT12 in small intestine, kidney, adipose tissue and skeletal muscle. Exercise alone does not modify GLUT12 expression, nevertheless exercise prevents the DHA-diminishing effect on GLUT12.

Metabolomics analysis of Actinosynnema pretiosum with improved AP-3 production by enhancing UDP-glucose biosynthesis

Ansamitocin P-3 (AP-3) shows strong anticancer effects and has used as a payload for antibody-drug conjugates. Our previous study have shown that although genetically engineered Actinosynnema pretiosum strains with enhanced UDP-glucose (UDPG) biosynthesis displayed improved AP-3 production compared to the wild-type strain, the increase in yield was far from meeting the industrial demand. In this study, comparative metabolomics analysis complemented with quantitative real-time PCR analysis was performed for the wild-type strain and two mutants (OpgmOugp, ΔzwfΔgnd) to identify possible metabolic bottlenecks and non-intuitive targets for further enhancement of AP-3 production. We observed that enhancing intracellular UDPG availability facilitated the accumulation of intracellular N-demethyl-AP-3 and AP-3, where the transporting of them outside the cell still needs to be developed. We also found that the UDPG biosynthesis was closely associated with the availability of fructose in the medium and a suitable fructose feeding strategy could promote the further improvement of AP-3 titer. In addition, pathway abundance analysis revealed that undesired fatty acid accumulation and down-regulation of amino acid metabolism may be unfavorable for ansamitocin biosynthesis in later stage of production. These results indicate that genetic modification of the UDPG biosynthetic pathways may have pleiotropic effects on AP-3 production. Efforts must be made to eliminate these newly identified metabolic bottlenecks to boost AP-3 production in A.pretiosum.

Untargeted Metabolic Pathway Analysis as an Effective Strategy to Connect Various Nanoparticle Properties to Nanoparticle-Induced Ecotoxicity

Elucidation of the relationships between nanoparticle properties and ecotoxicity is a fundamental issue for environmental applications and risk assessment of nanoparticles. However, effective strategies to connect the various properties of nanoparticles with their ecotoxicity remain largely unavailable. Herein, an untargeted metabolic pathway analysis was employed to investigate the environmental risk posed by 10 typical nanoparticles (AgNPs, CuNPs, FeNPs, ZnONPs, SiO2NPs, TiO2NPs, GO, GOQDs, SWCNTs, and C60) to rice (a staple food for half of the world's population). Downregulation of carbohydrate metabolism and upregulation of amino acid metabolism were the two dominant metabolic effects induced by all tested nanoparticles. Partial least-squares regression analysis indicated that a zerovalent metal and high specific surface area positively contributed to the downregulation of carbohydrate metabolism, indicating strong abiotic stress. In contrast, the carbon type, the presence of a spherical or sheet shape, and the absence of oxygen functional groups in the nanoparticles positively contributed to the upregulation of amino acid metabolism, indicating adaptation to abiotic stress. Moreover, network relationships among five properties of nanoparticles were established for these metabolic pathways. The results of the present study will aid in the understanding and prediction of environmental risks and in the design of environmentally friendly nanoparticles.

Potential therapeutic targets for lung repair during human ex vivo lung perfusion.

The ex vivo lung perfusion (EVLP) technique has been developed to assess the function of marginal donor lungs and has significantly increased donor lung utilisation. EVLP has also been explored as a platform for donor lung repair through injury-specific treatments such as antibiotics or fibrinolytics. We hypothesised that actively expressed pathways shared between transplantation and EVLP may reveal common mechanisms of injury and potential therapeutic targets for lung repair prior to transplantation. Retrospective transcriptomics analyses were performed with peripheral tissue biopsies from "donation after brain death" lungs, with 46 pre-/post-transplant pairs and 49 pre-/post-EVLP pairs. Pathway analysis was used to identify and compare the responses of donor lungs to transplantation and to EVLP. 22 pathways were enriched predominantly in transplantation, including upregulation of lymphocyte activation and cell death and downregulation of metabolism. Eight pathways were enriched predominantly in EVLP, including downregulation of leukocyte functions and upregulation of vascular processes. 27 pathways were commonly enriched, including activation of innate inflammation, cell death, heat stress and downregulation of metabolism and protein synthesis. Of the inflammatory clusters, Toll-like receptor/innate immune signal transduction adaptor signalling had the greatest number of nodes and was central to inflammation. These mechanisms have been previously speculated as major mechanisms of acute lung injury in animal models. EVLP and transplantation share common molecular features of injury including innate inflammation and cell death. Blocking these pathways during EVLP may allow for lung repair prior to transplantation.

Effect of Natural Cytokine Complex on Metabolism of Smooth Muscle Cells in Myocardial Arteries under Normal Conditions and during Hemodynamic Overload.

Histoenzymological methods were employed to examine the effects of systemically administered natural cytokine complex including IL-1, IL-2, IL-6, TNFα, MIF, and TGFβ on metabolism of smooth muscle cells in intramural myocardial arteries under physiological conditions and during acute hemodynamic overload of the heart. Natural cytokine complex markedly inhibited metabolism of vascular smooth muscle cells under control conditions and during acute experimental aortal stenosis. In vascular smooth muscle cells, deceleration of tricarboxylic acid cycle, redistribution of the fluxes in glycolytic cascade and its inhibition, down-regulation of oxidation of free fatty acids and their metabolites, and inhibition of the shuttle systems and biosynthetic processes were observed. Inhibition of metabolism in the vascular wall of myocardial arteries correlated with a decrease in their tone and could be partially determined by a decrease in contractile activity of smooth muscle cells. These findings do not exclude the involvement of other factors and mechanisms in down-regulation of metabolism in vascular myocytes in response to increased cytokines levels of in the blood, including their direct effect on biochemical processes in cells.

Skeletal Muscle Energetics and Mitochondrial Function Are Impaired Following 10 Days of Bed Rest in Older Adults.

BackgroundOlder adults exposed to periods of inactivity during hospitalization, illness, or injury lose muscle mass and strength. This, in turn, predisposes poor recovery of physical function upon reambulation and represents a significant health risk for older adults. Bed rest (BR) results in altered skeletal muscle fuel metabolism and loss of oxidative capacity that have recently been linked to the muscle atrophy program. Our primary objective was to explore the effects of BR on mitochondrial energetics in muscle from older adults. A secondary objective was to examine the effect of β-hydroxy-β-methylbuturate (HMB) supplementation on mitochondrial energetics.MethodsWe studied 20 older adults before and after a 10-day BR intervention, who consumed a complete oral nutritional supplement (ONS) with HMB (3.0 g/d HMB, n = 11) or without HMB (CON, n = 9). Percutaneous biopsies of the vastus lateralis were obtained to determine mitochondrial respiration and H2O2 emission in permeabilized muscle fibers along with markers of content. RNA sequencing and lipidomics analyses were also conducted.ResultsWe found a significant up-regulation of collagen synthesis and down-regulation of ribosome, oxidative metabolism and mitochondrial gene transcripts following BR in the CON group. Alterations to these gene transcripts were significantly blunted in the HMB group. Mitochondrial respiration and markers of content were both reduced and H2O2 emission was elevated in both groups following BR.ConclusionsIn summary, 10 days of BR in older adults causes a significant deterioration in mitochondrial energetics, while transcriptomic profiling revealed that some of these negative effects may be attenuated by an ONS containing HMB.

Spatial Organization of Expanding Bacterial Colonies Is Affected by Contact-Dependent Growth Inhibition.

SummaryIdentifying how microbes are able to manipulate, survive, and thrive in complex multispecies communities has expanded our understanding of how microbial ecosystems impact human health and the environment. The ability of bacteria to negatively affect neighbors, through explicit toxin delivery systems, provides them with an opportunity to manipulate the composition of growing microbial communities. Contact-dependent inhibition (CDI) systems (a Type Vb secretion system) are a distinct subset of competition systems whose contribution to shaping the development of spatially structured bacterial communities are yet to be fully understood. Here, we compare the impact of different CDI systems, at both the single-cell and population level, to determine the key drivers of CDI-mediated competition within spatially structured bacterial populations. Through an iterative approach using both an Escherichia coli experimental system and computational modeling, we show that CDI systems have subtle and system-specific effects at the single-cell level, generating single-cell-wide boundaries between CDI-expressing inhibitor cells and their neighboring targets. Despite the subtle effects of CDI at a single-cell level, CDI systems greatly diminished the ability of susceptible targets to expand their range during colony growth. The inoculum density of the population, together with the CDI system-specific variables of the speed of inhibition after contact and biological cost of CDI, strongly affects CDI-mediated competition. In contrast, the magnitude of the toxin-induced growth retardation of target cells only weakly impacts the composition of the population. Our work reveals how distinct CDI systems can differentially affect the composition and spatial arrangement of bacterial populations.

The Expression of Virulence Factors in Vibrio anguillarum Is Dually Regulated by Iron Levels and Temperature.

Vibrio anguillarum causes a hemorrhagic septicemia that affects cold- and warm-water adapted fish species. The main goal of this work was to determine the temperature-dependent changes in the virulence factors that could explain the virulence properties of V. anguillarum for fish cultivated at different temperatures. We have found that although the optimal growth temperature is around 25°C, the degree of virulence of V. anguillarum RV22 is higher at 15°C. To explain this result, an RNA-Seq analysis was performed to compare the whole transcriptome profile of V. anguillarum RV22 cultured under low-iron availability at either 25 or 15°C, which would mimic the conditions that V. anguillarum finds during colonization of fish cultivated at warm- or cold-water temperatures. The comparative analysis of transcriptomes at high- and low-iron conditions showed profound metabolic adaptations to grow under low iron. These changes were characterized by a down-regulation of the energetic metabolism and the induction of virulence-related factors like biosynthesis of LPS, production of hemolysins and lysozyme, membrane transport, heme uptake, or production of siderophores. However, the expression pattern of virulence factors under iron limitation showed interesting differences at warm and cold temperatures. Chemotaxis, motility, as well as the T6SS1 genes are expressed at higher levels at 25°C than at 15°C. By contrast, hemolysin RTX pore-forming toxin, T6SS2, and the genes associated with exopolysaccharides synthesis were preferentially expressed at 15°C. Notably, at this temperature, the siderophore piscibactin system was strongly up-regulated. In contrast, at 25°C, piscibactin genes were down-regulated and the vanchrobactin siderophore system seems to supply all the necessary iron to the cell. The results showed that V. anguillarum adjusts the expression of virulence factors responding to two environmental signals, iron levels and temperature. Thus, the relative relevance of each virulence factor for each fish species could vary depending on the water temperature. The results give clues about the physiological adaptations that allow V. anguillarum to cause infections in different fishes and could be relevant for vaccine development against fish vibriosis.

A phosphorylation-deficient mutant of retinoid X receptor α at Thr 167 alters fasting response and energy metabolism in mice

Retinoid X receptor α (RXRα) has a conserved phosphorylation motif at threonine 162 (humans) and threonine 167 (mice) within the DNA-binding domain. Here we have generated RXRα knock-in mice (RxrαT167A) bearing a single mutation of Thr 167 to alanine and examined the roles of Thr 167 in the regulation of energy metabolism within adipose, muscle, and liver tissues. RxrαT167A mice exhibited down-regulation of metabolic pathways converting glucose to fatty acids, such as acetyl-CoA carboxylase in the white adipose tissue (WAT) and ATP citrate lyase in the muscle. They also reduced gene expression for genes related to fatty acid catabolism and triglyceride synthesis in WAT and controlled heat factors such as adrenergic receptor β1 in muscles. In contrast, hepatic gluconeogenic pathways and synthetic pathways related to fatty acids remained unaffected by this mutation. Expression of multiple genes that were affected by the Thr 167 mutation in adipose tissue exhibited clear response to LG100268, a synthetic RXR agonist. Thus, the altered gene expression in mutant mice adipose appeared to be a direct effect of RXRα Thr 167 mutation and by some secondary effect of the mutation. Blood glucose levels remained normal in RxrαT167A during feeding, as observed with RXRα wild-type mice. However, RxrαT167A mice exhibited an attenuated decrease of blood glucose levels that occurred after fasting. This attenuation correlated with a concomitant down-regulation of lipid metabolism in WAT and was associated with RXRα phosphorylation at Thr 167. Thus, Thr 167 enabled RXRα to coordinate these three organs for regulation of energy metabolism and maintenance of glucose homeostasis.

Sex-specific hippocampal metabolic signatures at the onset of systemic inflammation with lipopolysaccharide in the APPswe/PS1dE9 mouse model of Alzheimer’s disease

Systemic inflammation enhances the risk and progression of Alzheimer’s disease (AD). Lipopolysaccharide (LPS), a potent pro-inflammatory endotoxin produced by the gut, is found in excess levels in AD where it associates with neurological hallmarks of pathology. Sex differences in susceptibility to inflammation and AD progression have been reported, but how this impacts on LPS responses remains under investigated. We previously reported in an APP/PS1 model of AD that systemic LPS administration rapidly altered hippocampal metabolism in males. Here, we used untargeted metabolomics to comprehensively identify hippocampal metabolic processes occurring at onset of systemic inflammation with LPS (100 µg/kg, i.v.) in APP/PS1 mice, at an early pathological stage, and investigated the sexual dimorphism in this response. Four hours after LPS administration, pathways regulating energy metabolism, immune and oxidative stress responses were simultaneously recruited in the hippocampi of 4.5-month-old mice with a more protective response in females despite their pro-inflammatory and pro-oxidant metabolic signature in the absence of immune stimulation. LPS induced comparable behavioural sickness responses in male and female wild-type and APP/PS1 mice and comparable activation of both the serotonin and nicotinamide pathways of tryptophan metabolism in their hippocampi. Elevations in N-methyl-2-pyridone-5-carboxamide, a major toxic metabolite of nicotinamide, correlated with behavioural sickness regardless of sex, as well as with the LPS-induced hypothermia seen in males. Males also exhibited a pro-inflammatory-like downregulation of pyruvate metabolism, exacerbated in APP/PS1 males, and methionine metabolism whereas females showed a greater cytokine response and anti-inflammatory-like downregulation of hippocampal methylglyoxal and methionine metabolism. Metabolic changes were not associated with morphological markers of immune cell activation suggesting that they constitute an early event in the development of LPS-induced neuroinflammation and AD exacerbation. These data suggest that the female hippocampus is more tolerant to acute systemic inflammation.

Nicotinamide Riboside Augments the Aged Human Skeletal Muscle NAD+ Metabolome and Induces Transcriptomic and Anti-inflammatory Signatures

SummaryNicotinamide adenine dinucleotide (NAD+) is modulated by conditions of metabolic stress and has been reported to decline with aging in preclinical models, but human data are sparse. Nicotinamide riboside (NR) supplementation ameliorates metabolic dysfunction in rodents. We aimed to establish whether oral NR supplementation in aged participants can increase the skeletal muscle NAD+ metabolome and if it can alter muscle mitochondrial bioenergetics. We supplemented 12 aged men with 1 g NR per day for 21 days in a placebo-controlled, randomized, double-blind, crossover trial. Targeted metabolomics showed that NR elevated the muscle NAD+ metabolome, evident by increased nicotinic acid adenine dinucleotide and nicotinamide clearance products. Muscle RNA sequencing revealed NR-mediated downregulation of energy metabolism and mitochondria pathways, without altering mitochondrial bioenergetics. NR also depressed levels of circulating inflammatory cytokines. Our data establish that oral NR is available to aged human muscle and identify anti-inflammatory effects of NR.

Abstract 331: Dynamic kinome targeting reveals kinases involved in acquired resistance to tyrosine kinase inhibitors in EGFR-driven glioblastomas

Abstract Glioblastoma (GBM) is a devastating primary brain tumor with limited treatment options. Extensive molecular characterization has revealed two particularly frequent mutations: CDKN2A deletion (50-60%) and EGFR (40-50%). EGFRvIII (~35%) is a constitutively active truncation mutant with exons 2-7 deleted. EGFR is a particularly attractive therapeutic target due to frequent activating mutations, such as EGFRvIII, and ready availability of multiple targeted inhibitors. Several EGFR tyrosine kinase inhibitors (TKI) have failed clinically, due in part to acquired resistance. To mechanistically examine this type of resistance, we used genetically-engineered mouse astrocytes harboring homozygous deletions of Cdkn2a, as well as EGFRvIII (CEv3). CEv3 astrocytes were made intrinsically resistant to the EGFR TKI gefitinib or erlotinib via long-term exposure, both in vitro and in vivo. We found that long-term gefitinib or erlotinib exposure conferred variable levels of cross resistance to a panel of second- and third-generation EGFR TKI (ΔIC50 1.12-36.1-fold), relative to non-resistant parent lines. We have previously shown that dynamic kinome reprogramming may be responsible for TKI resistance in glioblastoma. Therefore, we used a chemical proteomics method, multiplexed inhibitor beads and mass spectrometry (MIB-MS), to examine changes in the expressed and functional kinome, in both the presence or absence of one of several EGFR TKI known to penetrate the blood-brain barrier. Additionally, we performed RNA sequencing (RNA-seq) to inspect transcriptomic alterations in response to these drugs. RNA-seq showed that resistant CEv3 mouse astrocytes clustered separately from their non-resistant in vitro and in vivo counterparts. Acquired resistance also induced transcriptome alterations governing cellular metabolism, including upregulation of metabolic pathways and downregulation of RNA processing genes. Importantly, the kinase transcriptome was rewired, as 67 kinases were differentially expressed across parental and resistant cell lines (Q<0.001). Probing the dynamic kinome response to afatinib, an EGFR TKI, using RNA-seq identified two potential kinases involved in acute, adaptive resistance to afatinib, Bmx and Ntrk3. Integrated kinome profiling using RNA-seq and MIB-MS in murine models of GBM with defined mutational profiles provides a powerful framework to define novel therapeutic targets that could significantly alter current treatment paradigms. Citation Format: Abby Shelton, Erin Smithberger, Madison Butler, Alex Flores, Ryan Bash, Steve Angus, Noah Sciaky, Harshil Dhruv, Gary L. Johnson, Michael E. Berens, Frank Furnari, C. Ryan Miller. Dynamic kinome targeting reveals kinases involved in acquired resistance to tyrosine kinase inhibitors in EGFR-driven glioblastomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 331.