Enteric Methane Emissions from Dairy-Beef Steers Supplemented with the Essential Oil Blend Agolin Ruminant.

Agriculture is the largest source of methane globally, and enteric methane accounts for 32% of methane emissions globally. Dairy-beef is an increasingly important contributor to the beef industry. The objective of this study was to investigate if supplementation with a blend of essential oils (Agolin Ruminant) reduced enteric methane emissions from dairy-bred steers. Methane was measured from thirty-six Holstein Friesian steers (18 control and 18 treatment) in open-circuit respiration chambers, at three time-points relative to the introduction of Agolin Ruminant: (i) -3 (pre-additive introduction co-variate), (ii) 46 days after introduction, and (iii) 116 days after introduction. A significantly lower methane yield was observed in treated animals compared to control animals at both 46 days (p < 0.05) and 116 days (p < 0.01) after the introduction of Agolin Ruminant, although there was no difference in methane production (g/day). Control animals appeared to be more affected by isolation in respiration chambers than animals receiving Agolin Ruminant, as indicated by a significant reduction in dry matter intake by control animals in respiration chambers.

AMPK protects endothelial cells against HSV-1 replication via inhibition of mTORC1 and ACC1.

Herpes simplex virus type 1 (HSV-1) is a widespread contagious pathogen, mostly causing mild symptoms on the mucosal entry side. However, systemic distribution, in particular upon reactivation of the virus in immunocompromised patients, may trigger an innate immune response and induce damage of organs. In these conditions, HSV-1 may infect vascular endothelial cells, but little is known about the regulation of HSV-1 replication and possible defense mechanisms in these cells. The current study addresses the question of whether the host cell protein AMP-activated protein kinase (AMPK), an important metabolic sensor, can control HSV-1 replication in endothelial cells. We show that downregulation of the catalytic subunits AMPKα1 and/or AMPKα2 increased HSV-1 replication as monitored by TCID50 titrations, while a potent AMPK agonist, MK-8722, strongly inhibited it. MK-8722 induced a persistent phosphorylation of the AMPK downstream targets acetyl-CoA carboxylase (ACC) and the rapamycin-sensitive adaptor protein of mTOR (Raptor) and, related to this, impairment of ACC1-mediated lipid synthesis and the mechanistic target of the rapamycin complex-1 (mTORC1) pathway. Since blockade of mTOR by Torin-2 as well as downregulation of ACC1 by siRNA also decreased HSV-1 replication, MK-8722 is likely to exert its anti-viral effect via mTORC1 and ACC1 inhibition. Importantly, MK-8722 was able to reduce virus replication even when added after HSV-1. Together, our data highlight the importance of endothelial cells as host cells for HSV-1 replication upon systemic infection and identify AMPK, a metabolic host cell protein, as a potential target for antiviral strategies against HSV-1 infection and its severe consequences. IMPORTANCE Herpes simplex virus type 1 (HSV-1) is a common pathogen that causes blisters or cold sores in humans. It remains latent in infected individuals and can be reactivated multiple times. In adverse conditions, for instance, in immunocompromised patients, HSV-1 can lead to serious complications such as encephalitis, meningitis, or blindness. In these situations, infection of endothelial cells lining the surface of blood vessels may contribute to the manifestation of disease. Here, we describe the role of AMP-activated protein kinase (AMPK), a potent regulator of cellular energy metabolism, in HSV-1 replication in endothelial cells. While downregulation of AMPK potentiates HSV-1 replication, pharmacological AMPK activation inhibits it by limiting the availability of required host cell macromolecules such as proteins or fatty acids. These data highlight the role of metabolic host cell proteins as antiviral targets and reveal activation of endothelial AMPK as a potential strategy to protect from severe consequences of HSV-1 infection.

Modulation of milking performance, methane emissions, and rumen microbiome on dairy cows by dietary supplementation of a blend of essential oils.

Cattle represent a high contribution of the livestock's greenhouse gas emissions, mainly in the form of methane. Essential oils are a group of plant secondary metabolites obtained from volatile fractions of plants that have been shown to exert changes in the rumen fermentation and may alter feed efficiency and to reduce methane production. The objective of this study was to investigate the effect on rumen microbial population, CH4 emissions and milking performance of a mixture of essential oils (Agolin Ruminant, Switzerland) incorporated daily in the ration of dairy cattle. Forty Holstein cows (644 ± 63.5 kg of BW producing 41.2 ± 6.44 kg/d of milk with 190 ± 28.3 DIM) were divided into two treatments (n = 20) for 13 wk and housed in a single pen equipped with electronic feeding gates to control access to feed and monitor individual DM intake (DMI) on a daily basis. Treatments consisted of no supplementation (Control) or supplementation of 1 g/d of a blend of essential oils (BEOs) fed in the TMR. Individual milk production was recorded using electronic milk meters on a daily basis. Methane emissions were recorded using sniffers at the exit of the milking parlour. At day 64 of the study, a sample of rumen fluid was collected from 12 cows per treatment after the morning feeding using a stomach tube. There were no differences in DMI, milk yield, or milk composition between the two treatments. However, cows on BEO exhaled less CH4 (444 ± 12.5 l/d) than cows on Control (479 ± 12.5 l/d), and exhaled less (P < 0.05) CH4/kg of DM consumed (17.6 vs 20.1 ± 0.53 l/kg, respectively) from the first week of study, with no interaction with time, which suggests a fast action of BEO of CH4 emissions. Rumen relative abundance of Entodonium increased, and those of Fusobacteria, Chytridiomycota, Epidinium, and Mogibacterium decreased in BEO compared with Control cows. Supplementing 1 g/d of BEO reduces CH4 emissions on absolute terms (l/d) and diminishes the amount of CH4 produced by unit of DM consumed by cows relatively soon after the first supplementation, and the effect is sustained over time without impacting intake or milking performance.

Impact of essential oils on methane emissions, milk yield, and feed efficiency and resulting influence on the carbon footprint of dairy production systems.

Reducing CO2 emissions is one of the highest priorities in animal production. Regarding methane reduction, feed additives are of growing importance. As shown in a meta-analysis, the use of the essential oil (EO) blend Agolin Ruminant affects methane production per day (- 8.8%), milk yield (+ 4.1%), and feed efficiency (+ 4.4%). Building on these results, the present study investigated the effect of varying individual parameters on the carbon footprint of milk. The environmental and operational management system REPRO was applied to calculate the CO2 emissions. Calculation of CO2 emissions include enteric and storage-related CH4, storage-, and pasture-related N2O as well as direct and indirect energy expenditures. Three feed rations were created, differing in their basic feed components such as grass silage, corn silage, and pasture. Each feed ration was differentiated into three variants: variant 1 CON (no additive), variant 2 EO, and variant 3 (15% reduction of enteric methane compared to CON). Due to the reducing effect of EO on enteric methane production, a reduction potential of up to 6% could be calculated for all rations. Considering other variable parameters, such as the positive effects on ECM yield and feed efficiency, a GHG reduction potential of up to 10% can be achieved for the silage rations and almost 9% for the pasture ration. Modeling showed that indirect methane reduction strategies are important contributors to environmental impacts. Reduction of enteric methane emissions is fundamental, as they account for the largest share of GHG emissions from dairy production.

Oxidative Glucose Metabolism Promotes Senescence in Vascular Endothelial Cells.

Vascular aging is based on the development of endothelial dysfunction, which is thought to be promoted by senescent cells accumulating in aged tissues and is possibly affected by their environment via inflammatory mediators and oxidative stress. Senescence appears to be closely interlinked with changes in cell metabolism. Here, we describe an upregulation of both glycolytic and oxidative glucose metabolism in replicative senescent endothelial cells compared to young endothelial cells by employing metabolic profiling and glucose flux measurements and by analyzing the expression of key metabolic enzymes. Senescent cells exhibit higher glycolytic activity and lactate production together with an enhanced expression of lactate dehydrogenase A as well as increases in tricarboxylic acid cycle activity and mitochondrial respiration. The latter is likely due to the reduced expression of pyruvate dehydrogenase kinases (PDHKs) in senescent cells, which may lead to increased activity of the pyruvate dehydrogenase complex. Cellular and mitochondrial ATP production were elevated despite signs of mitochondrial dysfunction, such as an increased production of reactive oxygen species and extended mitochondrial mass. A shift from glycolytic to oxidative glucose metabolism induced by pharmacological inhibition of PDHKs in young endothelial cells resulted in premature senescence, suggesting that alterations in cellular glucose metabolism may act as a driving force for senescence in endothelial cells.

Structure-function analysis of the AMPK activator SC4 and identification of a potent pan AMPK activator.

The AMP-activated protein kinase (AMPK) αßγ heterotrimer is a primary cellular energy sensor and central regulator of energy homeostasis. Activating skeletal muscle AMPK with small molecule drugs improves glucose uptake and provides an opportunity for new strategies to treat type 2 diabetes and insulin resistance, with recent genetic and pharmacological studies indicating the α2ß2γ1 isoform combination as the heterotrimer complex primarily responsible. With the goal of developing α2ß2-specific activators, here we perform structure/function analysis of the 2-hydroxybiphenyl group of SC4, an activator with tendency for α2-selectivity that is also capable of potently activating ß2 complexes. Substitution of the LHS 2-hydroxyphenyl group with polar-substituted cyclohexene-based probes resulted in two AMPK agonists, MSG010 and MSG011, which did not display α2-selectivity when screened against a panel of AMPK complexes. By radiolabel kinase assay, MSG010 and MSG011 activated α2ß2γ1 AMPK with one order of magnitude greater potency than the pan AMPK activator MK-8722. A crystal structure of MSG011 complexed to AMPK α2ß1γ1 revealed a similar binding mode to SC4 and the potential importance of an interaction between the SC4 2-hydroxyl group and α2-Lys31 for directing α2-selectivity. MSG011 induced robust AMPK signalling in mouse primary hepatocytes and commonly used cell lines, and in most cases this occurred in the absence of changes in phosphorylation of the kinase activation loop residue α-Thr172, a classical marker of AMP-induced AMPK activity. These findings will guide future design of α2ß2-selective AMPK activators, that we hypothesise may avoid off-target complications associated with indiscriminate activation of AMPK throughout the body.

Mapping protein carboxymethylation sites provides insights into their role in proteostasis and cell proliferation.

Posttranslational mechanisms play a key role in modifying the abundance and function of cellular proteins. Among these, modification by advanced glycation end products has been shown to accumulate during aging and age-associated diseases but specific protein targets and functional consequences remain largely unexplored. Here, we devise a proteomic strategy to identify sites of carboxymethyllysine modification, one of the most abundant advanced glycation end products. We identify over 1000 sites of protein carboxymethylation in mouse and primary human cells treated with the glycating agent glyoxal. By using quantitative proteomics, we find that protein glycation triggers a proteotoxic response and indirectly affects the protein degradation machinery. In primary endothelial cells, we show that glyoxal induces cell cycle perturbation and that carboxymethyllysine modification reduces acetylation of tubulins and impairs microtubule dynamics. Our data demonstrate the relevance of carboxymethyllysine modification for cellular function and pinpoint specific protein networks that might become compromised during aging.

Circulating Bile Acids in Liver Failure Activate TGR5 and Induce Monocyte Dysfunction.

BACKGROUND & AIMS: Retention of bile acids in the blood is a hallmark of liver failure. Recent studies have shown that increased serum bile acid levels correlate with bacterial infection and increased mortality. However, the mechanisms by which circulating bile acids influence patient outcomes still are elusive. METHODS: Serum bile acid profiles in 33 critically ill patients with liver failure and their effects on Takeda G-protein-coupled receptor 5 (TGR5), an immunomodulatory receptor that is highly expressed in monocytes, were analyzed using tandem mass spectrometry, novel highly sensitive TGR5 bioluminescence resonance energy transfer using nanoluciferase (NanoBRET, Promega Corp, Madison, WI) technology, and in vitro assays with human monocytes. RESULTS: Twenty-two patients (67%) had serum bile acids that led to distinct TGR5 activation. These TGR5-activating serum bile acids severely compromised monocyte function. The release of proinflammatory cytokines (eg, tumor necrosis factor α or interleukin 6) in response to bacterial challenge was reduced significantly if monocytes were incubated with TGR5-activating serum bile acids from patients with liver failure. By contrast, serum bile acids from healthy volunteers did not influence cytokine release. Monocytes that did not express TGR5 were protected from the bile acid effects. TGR5-activating serum bile acids were a risk factor for a fatal outcome in patients with liver failure, independent of disease severity. CONCLUSIONS: Depending on their composition and quantity, serum bile acids in liver failure activate TGR5. TGR5 activation leads to monocyte dysfunction and correlates with mortality, independent of disease activity. This indicates an active role of TGR5 in liver failure. Therefore, TGR5 and bile acid metabolism might be promising targets for the treatment of immune dysfunction in liver failure.

Protein kinase A negatively regulates VEGF-induced AMPK activation by phosphorylating CaMKK2 at serine 495.

Activation of AMP-activated protein kinase (AMPK) in endothelial cells by vascular endothelial growth factor (VEGF) via the Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) represents a pro-angiogenic pathway, whose regulation and function is incompletely understood. This study investigates whether the VEGF/AMPK pathway is regulated by cAMP-mediated signalling. We show that cAMP elevation in endothelial cells by forskolin, an activator of the adenylate cyclase, and/or 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterases, triggers protein kinase A (PKA)-mediated phosphorylation of CaMKK2 (serine residues S495, S511) and AMPK (S487). Phosphorylation of CaMKK2 by PKA led to an inhibition of its activity as measured in CaMKK2 immunoprecipitates of forskolin/IBMX-treated cells. This inhibition was linked to phosphorylation of S495, since it was not seen in cells expressing a non-phosphorylatable CaMKK2 S495C mutant. Phosphorylation of S511 alone in these cells was not able to inhibit CaMKK2 activity. Moreover, phosphorylation of AMPK at S487 was not sufficient to inhibit VEGF-induced AMPK activation in cells, in which PKA-mediated CaMKK2 inhibition was prevented by expression of the CaMKK2 S495C mutant. cAMP elevation in endothelial cells reduced basal and VEGF-induced acetyl-CoA carboxylase (ACC) phosphorylation at S79 even if AMPK was not inhibited. Together, this study reveals a novel regulatory mechanism of VEGF-induced AMPK activation by cAMP/PKA, which may explain, in part, inhibitory effects of PKA on angiogenic sprouting and play a role in balancing pro- and anti-angiogenic mechanisms in order to ensure functional angiogenesis.

VEGF Triggers Transient Induction of Autophagy in Endothelial Cells via AMPKα1.

AMP-activated protein kinase (AMPK) is activated by vascular endothelial growth factor (VEGF) in endothelial cells and it is significantly involved in VEGF-induced angiogenesis. This study investigates whether the VEGF/AMPK pathway regulates autophagy in endothelial cells and whether this is linked to its pro-angiogenic role. We show that VEGF leads to AMPKα1-dependent phosphorylation of Unc-51-like kinase 1 (ULK1) at its serine residue 556 and to the subsequent phosphorylation of the ULK1 substrate ATG14. This triggers initiation of autophagy as shown by phosphorylation of ATG16L1 and conjugation of the microtubule-associated protein light chain 3B, which indicates autophagosome formation; this is followed by increased autophagic flux measured in the presence of bafilomycin A1 and by reduced expression of the autophagy substrate p62. VEGF-induced autophagy is transient and probably terminated by mechanistic target of rapamycin (mTOR), which is activated by VEGF in a delayed manner. We show that functional autophagy is required for VEGF-induced angiogenesis and may have specific functions in addition to maintaining homeostasis. In line with this, inhibition of autophagy impaired VEGF-mediated formation of the Notch intracellular domain, a critical regulator of angiogenesis. Our study characterizes autophagy induction as a pro-angiogenic function of the VEGF/AMPK pathway and suggests that timely activation of autophagy-initiating pathways may help to initiate angiogenesis.

Influence of sphingosine-1-phosphate signaling on HCMV replication in human embryonal lung fibroblasts.

The human cytomegalovirus (HCMV) is a common pathogen, which causes severe or even deadly diseases in immunocompromised patients. In addition, congenital HCMV infection represents a major health concern affecting especially the lung tissue of the susceptible individuals. Antivirals are a useful strategy to treat HCMV-caused diseases. However, all approved drugs target viral proteins but significant toxicity and an increasing resistance against these compounds have been observed. In infected cells, numerous host molecules have been identified to play important roles during HCMV replication. Among others, HCMV infection depends on the presence of bioactive sphingolipids. In this study, the role of sphingosine-1-phosphate (S1P) signaling in HCMV-infected human embryonal lung fibroblasts (HELF) was analyzed. Viral replication depended on the functional activity of sphingosine kinases (SK). During SK inhibition, addition of extracellular S1P restored HCMV replication. Moreover, neutralization of extracellular S1P by anti-S1P antibodies decreased HCMV replication as well. While the application of FTY720 as an functional antagonist of S1P receptor (S1PR)1,3-5 signaling did not reduce HCMV replication significantly, JTE-013, an inhibitor of S1PR2, decreased viral replication. Furthermore, inhibition of Rac-1 activity reduced HCMV replication, whereas inhibition of the Rac-1 effector protein Rac-1-activated kinase 1 (PAK1) had no influence. In general, targeting S1P-induced pathways, which are essential for a successful HCMV replication, may represent a valuable strategy to develop new antiviral drugs.

Studying the Role of AMPK in Angiogenesis.

The role of AMPK in angiogenesis can be studied using in vitro and in vivo assays. The endothelial spheroid assay is a robust three-dimensional in vitro test, which allows investigation of tubular morphogenesis by integrating cell-cell as well as cell-matrix interactions. The Matrigel plug assay validates the process of angiogenesis in vivo and allows studies in genetically modified mice. Here, we give a detailed description of both assays and their application in AMPK research.

GFAT1 phosphorylation by AMPK promotes VEGF-induced angiogenesis.

Activation of AMP-activated protein kinase (AMPK) in endothelial cells regulates energy homeostasis, stress protection and angiogenesis, but the underlying mechanisms are incompletely understood. Using a label-free phosphoproteomic analysis, we identified glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1) as an AMPK substrate. GFAT1 is the rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP) and as such controls the modification of proteins by O-linked ß-N-acetylglucosamine (O-GlcNAc). In the present study, we tested the hypothesis that AMPK controls O-GlcNAc levels and function of endothelial cells via GFAT1 phosphorylation using biochemical, pharmacological, genetic and in vitro angiogenesis approaches. Activation of AMPK in primary human endothelial cells by 5-aminoimidazole-4-carboxamide riboside (AICAR) or by vascular endothelial growth factor (VEGF) led to GFAT1 phosphorylation at serine 243. This effect was not seen when AMPK was down-regulated by siRNA. Upon AMPK activation, diminished GFAT activity and reduced O-GlcNAc levels were observed in endothelial cells containing wild-type (WT)-GFAT1 but not in cells expressing non-phosphorylatable S243A-GFAT1. Pharmacological inhibition or siRNA-mediated down-regulation of GFAT1 potentiated VEGF-induced sprouting, indicating that GFAT1 acts as a negative regulator of angiogenesis. In cells expressing S243A-GFAT1, VEGF-induced sprouting was reduced, suggesting that VEGF relieves the inhibitory action of GFAT1/HBP on angiogenesis via AMPK-mediated GFAT1 phosphorylation. Activation of GFAT1/HBP by high glucose led to impairment of vascular sprouting, whereas GFAT1 inhibition improved sprouting even if glucose level was high. Our findings provide novel mechanistic insights into the role of HBP in angiogenesis. They suggest that targeting AMPK in endothelium might help to ameliorate hyperglycaemia-induced vascular dysfunction associated with metabolic disorders.

Maternal diabetes promotes mTORC1 downstream signalling in rabbit preimplantation embryos.

The mammalian target of rapamycin complex 1 (mTORC1) is known to be a central cellular nutrient sensor and master regulator of protein metabolism; therefore, it is indispensable for normal embryonic development. We showed previously in a diabetic pregnancy that embryonic mTORC1 phosphorylation is increased in case of maternal hyperglycaemia and hypoinsulinaemia. Further, the preimplantation embryo is exposed to increased L-leucine levels during a diabetic pregnancy. To understand how mTOR signalling is regulated in preimplantation embryos, we examined consequences of L-leucine and glucose stimulation on mTORC1 signalling and downstream targets in in vitro cultured preimplantation rabbit blastocysts and in vivo. High levels of L-leucine and glucose lead to higher phosphorylation of mTORC1 and its downstream target ribosomal S6 kinase 1 (S6K1) in these embryos. Further, L-leucine supplementation resulted in higher embryonic expression of genes involved in cell cycle (cyclin D1; CCND1), translation initiation (eukaryotic translation initiation factor 4E; EIF4E), amino acid transport (large neutral amino acid transporter 2; Lat2: gene SLC7A8) and proliferation (proliferating cell nuclear antigen; PCNA) in a mTORC1-dependent manner. Phosphorylation of S6K1 and expression patterns of CCND1 and EIF4E were increased in embryos from diabetic rabbits, while the expression of proliferation marker PCNA was decreased. In these embryos, protein synthesis was increased and autophagic activity was decreased. We conclude that mammalian preimplantation embryos sense changes in nutrient supply via mTORC1 signalling. Therefore, mTORC1 may be a decisive mediator of metabolic programming in a diabetic pregnancy.

Activation of AMP-activated protein kinase by vascular endothelial growth factor mediates endothelial angiogenesis independently of nitric-oxide synthase.

AMP-activated protein kinase (AMPK) is a sensor of cellular energy state and a regulator of cellular homeostasis. In endothelial cells, AMPK is stimulated via the upstream kinases LKB1 and Ca(2+)/calmodulin-dependent protein kinase kinase beta (CaMKKbeta). Previously, AMPK has been reported to activate endothelial nitric-oxide synthase (eNOS). Using genetic and pharmacological approaches, we show that vascular endothelial growth factor (VEGF) stimulates AMPK in human and mice endothelial cells via CaMKKbeta. VEGF-induced AMPK activation is potentiated under conditions of energy deprivation induced by 2-deoxyglucose. To investigate the role of AMPK in endothelial function, CaMKKbeta, AMPKalpha1, or AMPKalpha2 was down-regulated by RNA interference, and studies in AMPKalpha1(-/-) mice were performed. We demonstrate that AMPK does not mediate eNOS phosphorylation at serine residue 1177 or 633, NO- dependent cGMP generation, or Akt phosphorylation in response to VEGF. Using inhibitors of eNOS or soluble guanylyl cyclase and small interfering RNA against eNOS, we show that NO does not act upstream of AMPK. Taken together, these data indicate that VEGF-stimulated AMPK and eNOS pathways act independently of each other. However, acetyl-CoA carboxylase, a key enzyme in the regulation of fatty acid oxidation, was phosphorylated in response to VEGF in an AMPKalpha1- and AMPKalpha2-dependent manner. Our results show that AMPKalpha1 plays an essential role in VEGF-induced angiogenesis in vitro (tube formation and sprouting from spheroids) and in vivo (Matrigel plug assay). In contrast, AMPKalpha2 was not involved in VEGF-triggered sprouting. The data suggest that AMPKalpha1 promotes VEGF-induced angiogenesis independently of eNOS, possibly by providing energy via inhibition of acetyl-CoA carboxylase.