Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis

Impaired proinsulin-to-insulin processing in pancreatic β-cells is a key defective step in both type 1 diabetes and type 2 diabetes (T2D) (refs. $^{1}$$^{,}$$^{2}$), but the mechanisms involved remain to be defined. Altered metabolism of sphingolipids (SLs) has been linked to development of obesity, type 1 diabetes and T2D (refs. $^{3-8}$); nonetheless, the role of specific SL species in β-cell function and demise is unclear. Here we define the lipid signature of T2D-associated β-cell failure, including an imbalance of specific very-long-chain SLs and long-chain SLs. β-cell-specific ablation of CerS2, the enzyme necessary for generation of very-long-chain SLs, selectively reduces insulin content, impairs insulin secretion and disturbs systemic glucose tolerance in multiple complementary models. In contrast, ablation of long-chain-SL-synthesizing enzymes has no effect on insulin content. By quantitatively defining the SL-protein interactome, we reveal that CerS2 ablation affects SL binding to several endoplasmic reticulum-Golgi transport proteins, including Tmed2, which we define as an endogenous regulator of the essential proinsulin processing enzyme Pcsk1. Our study uncovers roles for specific SL subtypes and SL-binding proteins in β-cell function and T2D-associated β-cell failure

Comprehensive analysis of metabolic sensitivity of 1,4‐butanediol producing Escherichia coli toward substrate and oxygen availability

Nowadays, chemical production of 1,4‐butanediol is supplemented by biotechnological processes using a genetically modified Escherichia coli strain, which is an industrial showcase of successful application of metabolic engineering. However, large scale bioprocess performance can be affected by presence of physical and chemical gradients in bioreactors which are a consequence of imperfect mixing and limited oxygen transfer. Hence, upscaling comes along with local and time dependent fluctuations of cultivation conditions. This study emphasizes on scale‐up related effects of microbial 1,4‐butanediol production by comprehensive bioprocess characterization in lab scale. Due to metabolic network constraints 1,4‐butanediol formation takes place under oxygen limited microaerobic conditions, which can be hardly realized in large scale bioreactor. The purpose of this study was to assess the extent to which substrate and oxygen availability influence the productivity. It was found, that the substrate specific product yield and the production rate are higher under substrate excess than under substrate limitation. Furthermore, the level of oxygen supply within microaerobic conditions revealed strong effects on product and by‐product formation. Under strong oxygen deprivation nearly 30% of the consumed carbon is converted into 1,4‐butanediol, whereas an increase in oxygen supply results in 1,4‐butanediol reduction of 77%. Strikingly, increasing oxygen availability leads to strong increase of main by‐product acetate as well as doubled carbon dioxide formation. The study provides clear evidence that scale‐up of microaerobic bioprocesses constitute a substantial challenge. Although oxygen is strictly required for product formation, the data give clear evidence that terms of anaerobic and especially aerobic conditions strongly interfere with 1,4‐butanediol production

Comprehensive analysis of metabolic sensitivity of 1,4-butanediol producing Escherichia coli toward substrate and oxygen availability

Nowadays, chemical production of 1,4‐butanediol is supplemented by biotechnological processes using a genetically modified Escherichia coli strain, which is an industrial showcase of successful application of metabolic engineering. However, large scale bioprocess performance can be affected by presence of physical and chemical gradients in bioreactors which are a consequence of imperfect mixing and limited oxygen transfer. Hence, upscaling comes along with local and time dependent fluctuations of cultivation conditions. This study emphasizes on scale‐up related effects of microbial 1,4‐butanediol production by comprehensive bioprocess characterization in lab scale. Due to metabolic network constraints 1,4‐butanediol formation takes place under oxygen limited microaerobic conditions, which can be hardly realized in large scale bioreactor. The purpose of this study was to assess the extent to which substrate and oxygen availability influence the productivity. It was found, that the substrate specific product yield and the production rate are higher under substrate excess than under substrate limitation. Furthermore, the level of oxygen supply within microaerobic conditions revealed strong effects on product and by‐product formation. Under strong oxygen deprivation nearly 30% of the consumed carbon is converted into 1,4‐butanediol, whereas an increase in oxygen supply results in 1,4‐butanediol reduction of 77%. Strikingly, increasing oxygen availability leads to strong increase of main by‐product acetate as well as doubled carbon dioxide formation. The study provides clear evidence that scale‐up of microaerobic bioprocesses constitute a substantial challenge. Although oxygen is strictly required for product formation, the data give clear evidence that terms of anaerobic and especially aerobic conditions strongly interfere with 1,4‐butanediol production

Inhibition of 2-Oxoglutarate Dehydrogenase in Potato Tuber Suggests the Enzyme Is Limiting for Respiration and Confirms Its Importance in Nitrogen Assimilation1[W][OA]

The 2-oxoglutarate dehydrogenase complex constitutes a mitochondrially localized tricarboxylic acid cycle multienzyme system responsible for the conversion of 2-oxoglutarate to succinyl-coenzyme A concomitant with NAD+ reduction. Although regulatory mechanisms of plant enzyme complexes have been characterized in vitro, little is known concerning their role in plant metabolism in situ. This issue has recently been addressed at the cellular level in nonplant systems via the use of specific phosphonate inhibitors of the enzyme. Here, we describe the application of these inhibitors for the functional analysis of the potato (Solanum tuberosum) tuber 2-oxoglutarate dehydrogenase complex. In vitro experiments revealed that succinyl phosphonate (SP) and a carboxy ethyl ester of SP are slow-binding inhibitors of the 2-oxoglutarate dehydrogenase complex, displaying greater inhibitory effects than a diethyl ester of SP, a phosphono ethyl ester of SP, or a triethyl ester of SP. Incubation of potato tuber slices with the inhibitors revealed that they were adequately taken up by the tissue and produced the anticipated effects on the in situ enzyme activity. In order to assess the metabolic consequences of the 2-oxoglutarate dehydrogenase complex inhibition, we evaluated the levels of a broad range of primary metabolites using an established gas chromatography-mass spectrometry method. We additionally analyzed the rate of respiration in both tuber discs and isolated mitochondria. Finally, we evaluated the metabolic fate of radiolabeled acetate, 2-oxoglutarate or glucose, and 13C-labeled pyruvate and glutamate following incubation of tuber discs in the presence or absence of either SP or the carboxy ethyl ester of SP. The data obtained are discussed in the context of the roles of the 2-oxoglutarate dehydrogenase complex in respiration and carbon-nitrogen interactions

Metabolomic profile and nucleoside composition of Cordyceps nidus sp. nov. (Cordycipitaceae): A new source of active compounds.

Cordyceps sensu lato is a genus of arthropod-pathogenic fungi, which have been used traditionally as medicinal in Asia. Within the genus, Ophiocordyceps sinensis is the most coveted and expensive species in China. Nevertheless, harvesting wild specimens has become a challenge given that natural populations of the fungus are decreasing and because large-scale culture of it has not yet been achieved. The worldwide demand for products derived from cultivable fungal species with medicinal properties has increased recently. In this study, we propose a new species, Cordyceps nidus, which parasitizes underground nests of trapdoor spiders. This species is phylogenetically related to Cordyceps militaris, Cordyceps pruinosa, and a sibling species of Cordyceps caloceroides. It is found in tropical rainforests from Bolivia, Brazil, Colombia and Ecuador. We also investigated the medicinal potential of this fungus based on its biochemical properties when grown on four different culture media. The metabolic profile particularly that of nucleosides, in polar and non-polar extracts was determined by UPLC, and then correlated to their antimicrobial activity and total phenolic content. The metabolome showed a high and significant dependency on the substrate used for fungal growth. The mass intensities of nucleosides and derivative compounds were higher in natural culture media in comparison to artificial culture media. Among these compounds, cordycepin was the predominant, showing the potential use of this species as an alternative to O. sinensis. Furthermore, methanol fractions showed antimicrobial activity against gram-positive bacteria, and less than 3.00 mg of gallic acid equivalents per g of dried extract were obtained when assessing its total phenolic content by modified Folin-Ciocalteu method. The presence of polyphenols opens the possibility of further exploring the antioxidant capacity and the conditions that may enhance this characteristic. The metabolic composition and biochemical activity indicate potential use of C. nidus in pharmaceutical applications

An Integrated Genomics Approach to Define Niche Establishment by Rhodococcus fascians1[C][W][OA]

Rhodococcus fascians is a Gram-positive phytopathogen that induces shooty hyperplasia on its hosts through the secretion of cytokinins. Global transcriptomics using microarrays combined with profiling of primary metabolites on infected Arabidopsis (Arabidopsis thaliana) plants revealed that this actinomycete modulated pathways to convert its host into a niche. The transcript data demonstrated that R. fascians leaves a very characteristic mark on Arabidopsis with a pronounced cytokinin response illustrated by the activation of cytokinin perception, signal transduction, and homeostasis. The microarray data further suggested active suppression of an oxidative burst during the R. fascians pathology, and comparison with publicly available transcript data sets implied a central role for auxin in the prevention of plant defense activation. Gene Ontology categorization of the differentially expressed genes hinted at a significant impact of infection on the primary metabolism of the host, which was confirmed by subsequent metabolite profiling. The much higher levels of sugars and amino acids in infected plants are presumably accessed by the bacteria as carbon and nitrogen sources to support epiphytic and endophytic colonization. Hexoses, accumulating from a significantly increased invertase activity, possibly inhibited the expression of photosynthesis genes and photosynthetic activity in infected leaves. Altogether, these changes are indicative of sink development in symptomatic tissues. The metabolomics data furthermore point to the possible occurrence of secondary signaling during the interaction, which might contribute to symptom development. These data are placed in the context of regulation of bacterial virulence gene expression, suppression of defense, infection phenotype, and niche establishment

Fluctuating Light Interacts with Time of Day and Leaf Development Stage to Reprogram Gene Expression

Natural-light environments are highly variable. Flexible adjustment between light energy utilization and photoprotection is therefore of vital importance for plant performance and fitness in the field. Short-term reactions to changing light intensity are triggered inside chloroplasts and leaves within seconds to minutes, whereas long-term adjustments proceed over hours and days, integrating multiple signals. While the mechanisms of long-term acclimation to light intensity have been studied by changing constant growth light intensity during the day, responses to fluctuating growth light intensity have rarely been inspected in detail. We performed transcriptome profiling in Arabidopsis (Arabidopsis thaliana) leaves to investigate long-term gene expression responses to fluctuating light (FL). In particular, we examined whether responses differ between young and mature leaves or between morning and at the end of the day. Our results highlight global reprogramming of gene expression under FL, including that of genes related to photoprotection, photosynthesis and photorespiration and to pigment, prenylquinone and vitamin metabolism. The FL-induced changes in gene expression varied between young and mature leaves at the same time point, and between the same leaves in the morning and at the end of the day, indicating interactions of FL acclimation with leaf development stage and time of day. Only 46 genes were up- or downregulated in both young and mature leaves at both time points. Combined analyses of gene co-expression and cis-elements pointed to a role of the circadian clock and light in coordinating the acclimatory responses of functionally related genes. Our results also suggest a possible crosstalk between FL acclimation and systemic acquired resistance-like gene expression in young leaves

Mass intensities of nucleosides.

<p><b>A)</b> cordycepin, <b>B)</b> adenosine, <b>C)</b> inosine, <b>D)</b> guanosine, and <b>E)</b> uridine extracted from mycelium of <i>C</i>. <i>nidus</i> grown on different culture media. Methanol (black) and water (white) extracts. Statistical significance for alpha = 0.05: a. 9.26 10⁻⁹, b. 1.32 10⁻⁷, c. 5.83 10⁻¹¹, d. 4.11 10⁻¹¹, e. 3.72 10⁻³, f. 2.85 10⁻³, g. 1.64 10⁻¹⁴, h. 2.39 10⁻¹², i. 1.44 10⁻¹⁴.</p

Phylogenetic relationships inferred from a Maximum Likelihood analysis of <i>Cordyceps</i> species which parasitize spiders.

<p>Combined data set of SSU, LSU, TEF, RPB1, and RPB2 nuclear loci. Sequences from <i>Verticillium dahlie</i> and <i>Glomerella cingulata</i> were used as outgroups. Numbers above branches indicate Bootstrap support (BS) and below posterior probability support in Bayesian analyses (PP). Bionectriaceae, Nectriaceae, Hypocreaceae, Clavicipitaceae, Ophiocordycipitaceae are collapsed to emphasize of Cordycipitaceae.</p