Carbon monoxide exposure activates ULK1 via AMPK phosphorylation in murine embryonic fibroblasts.

Carbon monoxide (CO) is endogenously produced upon degradation of heme by heme oxygenases (HOs) and is suggested to act as a gaseous signaling molecule. The expression of HO-1 is triggered by the Nrf2-Keap1 signaling pathway which responds to exogenous stress signals and dietary constituents such as flavonoids and glucosinolates or reactive metabolic intermediates like 4-hydroxynonenal. Endogenous CO affects energy metabolism, regulates the utilization of glucose and addresses CYP450 enzymes. Using the CO releasing molecule-401 (CORM-401), we studied the effect of endogenous CO on ATP synthesis, AMP-signaling and activation of the AMPK pathway in cell culture. Upon exposure of cells to CORM-401, the mitochondrial ATP production rate was significantly decreased (P=0.007) to about 50%, while glycolytic ATP synthesis was unchanged (P=0.489). Total ATP levels were less affected as determined by mass spectrometry. Instead, levels of ADP and AMP were elevated following CORM-401 exposure by about two- (P=0.022) and four-fold (P=0.012) compared to control, respectively. Increased concentrations of AMP activate AMPK which was demonstrated by a 10 to 15-fold increased phosphorylation of Thr172 of the α-subunit of AMPK (P=0.025). A downstream target of AMPK is the kinase ULK1 which triggers autophagic and mitophagic processes. Activation of ULK1 after CO exposure was proven by a 3 to 5-fold elevated phosphorylation of ULK1 at Ser555 (P=0.004). The present data suggest that production of endogenous CO leads to increasing amounts of AMP which mediates AMPK-dependent downstream effects and likely triggers autophagic processes. Since dietary constituents and their metabolites induce the expression of the CO producing enzyme HO-1, CO signaling may also be involved in the cellular response to nutritional factors.

Ammonia inhibits energy metabolism in astrocytes in a rapid and glutamate dehydrogenase 2-dependent manner.

Astrocyte dysfunction is a primary factor in hepatic encephalopathy (HE) impairing neuronal activity under hyperammonemia. In particular, the early events causing ammonia-induced toxicity to astrocytes are not well understood. Using established cellular HE models, we show that mitochondria rapidly undergo fragmentation in a reversible manner upon hyperammonemia. Further, in our analyses, within a timescale of minutes, mitochondrial respiration and glycolysis were hampered, which occurred in a pH-independent manner. Using metabolomics, an accumulation of glucose and numerous amino acids, including branched chain amino acids, was observed. Metabolomic tracking of 15N-labeled ammonia showed rapid incorporation of 15N into glutamate and glutamate-derived amino acids. Downregulating human GLUD2 [encoding mitochondrial glutamate dehydrogenase 2 (GDH2)], inhibiting GDH2 activity by SIRT4 overexpression, and supplementing cells with glutamate or glutamine alleviated ammonia-induced inhibition of mitochondrial respiration. Metabolomic tracking of 13C-glutamine showed that hyperammonemia can inhibit anaplerosis of tricarboxylic acid (TCA) cycle intermediates. Contrary to its classical anaplerotic role, we show that, under hyperammonemia, GDH2 catalyzes the removal of ammonia by reductive amination of α-ketoglutarate, which efficiently and rapidly inhibits the TCA cycle. Overall, we propose a critical GDH2-dependent mechanism in HE models that helps to remove ammonia, but also impairs energy metabolism in mitochondria rapidly.

Corrigendum: Knock-Down of the Phosphoserine Phosphatase Gene Effects Rather N- Than S-Metabolism in Arabidopsis thaliana.

[This corrects the article DOI: 10.3389/fpls.2018.01830.].

The Photorespiratory BOU Gene Mutation Alters Sulfur Assimilation and Its Crosstalk With Carbon and Nitrogen Metabolism in Arabidopsis thaliana.

This study was aimed at elucidating the significance of photorespiratory serine (Ser) production for cysteine (Cys) biosynthesis. For this purpose, sulfur (S) metabolism and its crosstalk with nitrogen (N) and carbon (C) metabolism were analyzed in wildtype Arabidopsis and its photorespiratory bou-2 mutant with impaired glycine decarboxylase (GDC) activity. Foliar glycine and Ser contents were enhanced in the mutant at day and night. The high Ser levels in the mutant cannot be explained by transcript abundances of genes of the photorespiratory pathway or two alternative pathways of Ser biosynthesis. Despite enhanced foliar Ser, reduced GDC activity mediated a decline in sulfur flux into major sulfur pools in the mutant, as a result of deregulation of genes of sulfur reduction and assimilation. Still, foliar Cys and glutathione contents in the mutant were enhanced. The use of Cys for methionine and glucosinolates synthesis was reduced in the mutant. Reduced GDC activity in the mutant downregulated Calvin Cycle and nitrogen assimilation genes, upregulated key enzymes of glycolysis and the tricarboxylic acid (TCA) pathway and modified accumulation of sugars and TCA intermediates. Thus, photorespiratory Ser production can be replaced by other metabolic Ser sources, but this replacement deregulates the cross-talk between S, N, and C metabolism.

Knock-Down of the Phosphoserine Phosphatase Gene Effects Rather N- Than S-Metabolism in Arabidopsis thaliana.

The aim of present study was to elucidate the significance of the phosphorylated pathway of Ser production for Cys biosynthesis in leaves at day and night and upon cadmium (Cd) exposure. For this purpose, Arabidopsis wildtype plants as control and its psp mutant knocked-down in phosphoserine phosphatase (PSP) were used to test if (i) photorespiratory Ser is the dominant precursor of Cys synthesis in autotrophic tissue in the light, (ii) the phosphorylated pathway of Ser production can take over Ser biosynthesis in leaves at night, and (iii) Cd exposure stimulates Cys and glutathione (GSH) biosynthesis and effects the crosstalk of S and N metabolism, irrespective of the Ser source. Glycine (Gly) and Ser contents were not affected by reduction of the psp transcript level confirming that the photorespiratory pathway is the main route of Ser synthesis. The reduction of the PSP transcript level in the mutant did not affect day/night regulation of sulfur fluxes while day/night fluctuation of sulfur metabolite amounts were no longer observed, presumably due to slower turnover of sulfur metabolites in the mutant. Enhanced contents of non-protein thiols in both genotypes and of GSH only in the psp mutant were observed upon Cd treatment. Mutation of the phosphorylated pathway of Ser biosynthesis caused an accumulation of alanine, aspartate, lysine and a decrease of branched-chain amino acids. Knock-down of the PSP gene induced additional defense mechanisms against Cd toxicity that differ from those of WT plants.