Regulierung endothelialer Funktionen durch Autophagie und den AMPK/Autophagie-Signalweg

Endothelzellen bilden die innere Schicht von Blutgefäßen. Sie sind für die Aufrechterhaltung der vaskulären Homöostase wichtig und vermitteln Angiogenese, die durch Wachstumsfaktoren wie VEGF induziert wird und die Vaskularisierung von Geweben und Organen sichert. Voraussetzung für die Gewährleistung dieser Funktionen sind protektive Mechanismen, die Endothelzellen vor schädigenden Einflüssen schützen. Ein solcher Mechanismus ist Autophagie, ein lysosomaler Abbauweg, durch den alte und geschädigte Proteine und Organellen abgebaut werden, um so die Homöostase der Zellen aufrecht zu erhalten. Es konnte gezeigt werden, dass Endothelzellen unter basalen Bedingungen einen ausgeprägten Autophagie-Flux aufweisen, der für den Schutz vor oxidativem und inflammatorischem Stress und für Zellfunktionen wie Proliferation, Überleben und Angiogenese wichtig ist. Darüber hinaus konnte gezeigt werden, dass eine pharmakologische Aktivierung der AMPK, einer wichtigen Kinase zur Regulierung von Metabolismus und Zellschutzfunktionen, zu einem Anstieg von Autophagie-Parametern führt. VEGF als physiologischer AMPK-Aktivator führt ebenfalls zu einer Stimulierung der Autophagie. Die Aktivierung der Autophagie wird bei allen untersuchten Zugängen spezifisch durch die AMPKα1-Isoform vermittelt. Der VEGF/AMPKα1/Autophagie-Signalweg ist für den Prozess der Angiogenese von essentieller Bedeutung, da VEGF in Zellen mit gehemmter AMPKα1- oder Autophagie-Aktivität kaum mehr in der Lage ist, Angiogenese zu induzieren. Diese Daten geben damit neue Einblicke in die Regulierung der VEGF-stimulierten Angiogenese und charakterisieren den VEGF/AMPKα1/Autophagie-Signalweg als einen neuen proangiogenen Mechanismus. Insgesamt dienen die Ergebnisse dieser Arbeit dazu, die Regulation von Endothelzellfunktionen besser zu verstehen und damit die Basis für gezielte Maßnahmen zu liefern, die das Auftreten einer Endotheldysfunktion verhindern oder reduzieren können

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) αβγ 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 α2β2γ1 isoform combination as the heterotrimer complex primarily responsible. With the goal of developing α2β2-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 β2 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 α2β2γ1 AMPK with one order of magnitude greater potency than the pan AMPK activator MK-8722. A crystal structure of MSG011 complexed to AMPK α2β1γ1 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 α2β2-selective AMPK activators, that we hypothesise may avoid off-target complications associated with indiscriminate activation of AMPK throughout the body

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

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

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 pregnanc

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 β-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