Association of dietary patterns with diabetes-related comorbidities varies among diabetes endotypes.

BACKGROUND AND AIMS: Differences of dietary pattern adherence across the novel diabetes endotypes are unknown. This study assessed adherence to pre-specified dietary patterns and their associations with cardiovascular risk factors, kidney function, and neuropathy among diabetes endotypes. METHODS AND RESULTS: The cross-sectional analysis included 765 individuals with recent-onset (67 %) and prevalent diabetes (33 %) from the German Diabetes Study (GDS) allocated into severe autoimmune diabetes (SAID, 35 %), severe insulin-deficient diabetes (SIDD, 3 %), severe insulin-resistant diabetes (SIRD, 5 %), mild obesity-related diabetes (MOD, 28 %), and mild age-related diabetes (MARD, 29 %). Adherence to a Mediterranean diet score (MDS), Dietary Approaches to Stop Hypertension (DASH) score, overall plant-based diet (PDI), healthful (hPDI) and unhealthful plant-based diet index (uPDI) was derived from a food frequency questionnaire and associated with cardiovascular risk factors, kidney function, and neuropathy using multivariable linear regression analysis. Differences in dietary pattern adherence between endotypes were assessed using generalized mixed models. People with MARD showed the highest, those with SIDD and MOD the lowest adherence to the hPDI. Adherence to the MDS, DASH, overall PDI, and hPDI was inversely associated with high-sensitivity C-reactive protein (hsCRP) among people with MARD (ß (95%CI): -9.18 % (-15.61; -2.26); -13.61 % (-24.17; -1.58); -19.15 % (-34.28; -0.53); -16.10 % (-28.81; -1.12), respectively). Adherence to the PDIs was associated with LDL cholesterol among people with SAID, SIRD, and MOD. CONCLUSIONS: Minor differences in dietary pattern adherence (in particular for hPDI) and associations with markers of diabetes-related complications (e.g. hsCRP) were observed between endotypes. So far, evidence is insufficient to derive endotype-specific dietary recommendations. TRIAL REGISTRATION: Clinicaltrials.gov: NCT01055093.

Association of thyroid function with non-alcoholic fatty liver disease in recent-onset diabetes.

BACKGROUND AND AIMS: Non-alcoholic fatty liver disease (NAFLD) has been linked to type 2 diabetes (T2D), but also to hypothyroidism. Nevertheless, the relationship between thyroid function and NAFLD in diabetes is less clear. This study investigated associations between free thyroxine (fT4) or thyroid-stimulating hormone (TSH) and NAFLD in recent-onset diabetes. METHODS: Participants with recent-onset type 1 diabetes (T1D, n = 358), T2D (n = 596) or without diabetes (CON, n = 175) of the German Diabetes Study (GDS), a prospective longitudinal cohort study, underwent Botnia clamp tests and assessment of fT4, TSH, fatty liver index (FLI) and in a representative subcohort 1 H-magnetic resonance spectroscopy. RESULTS: First, fT4 levels were similar between T1D and T2D (p = .55), but higher than in CON (T1D: p < .01; T2D: p < .001), while TSH concentrations were not different between all groups. Next, fT4 correlated negatively with FLI and positively with insulin sensitivity only in T2D (ß = -.110, p < .01; ß = .126, p < .05), specifically in males (ß = -.117, p < .05; ß = .162; p < .01) upon adjustments for age, sex and BMI. However, correlations between fT4 and FLI lost statistical significance after adjustment for insulin sensitivity (T2D: ß = -.021, p = 0.67; males with T2D: ß = -.033; p = .56). TSH was associated positively with FLI only in male T2D before (ß = .116, p < .05), but not after adjustments for age and BMI (ß = .052; p = .30). CONCLUSIONS: Steatosis risk correlates with lower thyroid function in T2D, which is mediated by insulin resistance and body mass, specifically in men, whereas no such relationship is present in T1D.

Precise glucose measurement in sodium fluoride-citrate plasma affects estimates of prevalence in diabetes and prediabetes.

OBJECTIVES: Estimates of glucose concentrations vary among types of blood samples, which impact on the assessment of diabetes prevalence. Guidelines recommend a conversion factor to calculate plasma glucose from measurements of glucose in whole blood. The American Diabetes Association recommends the use of blood drawing tubes containing sodium fluoride (NaF) and citrate, which have not yet been evaluated regarding possible differences in glucose concentration and conversion factors. Thus, we compared glucose measurements in NaF-citrate plasma and venous whole blood and estimated the impact of differences on diabetes and prediabetes prevalence. METHODS: Glucose differences were calculated by Bland-Altman analysis with pairwise comparison of glucose measurements from whole blood and NaF-citrate plasma (n=578) in clinical studies of the German Diabetes Center. Subsequently, we computed the impact of the glucose difference on diabetes and prediabetes prevalence in the population-based National Health and Nutrition Examination Survey (NHANES). RESULTS: Even upon conversion of whole blood to plasma glucose concentrations using the recommended conversion factor, mean glucose concentration difference remained 4.72 % higher in NaF-citrate plasma. Applying the higher glucose estimates, increases the population-based diabetes and prediabetes prevalence by 13.67 and 33.97 % or more than 7.2 and 13 million people in NHANES, respectively. Additional economic burden could be about 20 $ billion per year due to undiagnosed diabetes. CONCLUSIONS: The recommended conversion factor is not valid for NaF-citrate plasma. Systematic bias of glucose measurements due to sampling type leads to clinically relevant higher estimates of diabetes and prediabetes prevalence.

Analysis of type 2 diabetes heterogeneity with a tree-like representation: insights from the prospective German Diabetes Study and the LURIC cohort.

BACKGROUND: Heterogeneity in type 2 diabetes can be represented by a tree-like graph structure by use of reversed graph-embedded dimensionality reduction. We aimed to examine whether this approach can be used to stratify key pathophysiological components and diabetes-related complications during longitudinal follow-up of individuals with recent-onset type 2 diabetes. METHODS: For this cohort analysis, 927 participants aged 18-69 years from the German Diabetes Study (GDS) with recent-onset type 2 diabetes were mapped onto a previously developed two-dimensional tree based on nine simple clinical and laboratory variables, residualised for age and sex. Insulin sensitivity was assessed by a hyperinsulinaemic-euglycaemic clamp, insulin secretion was assessed by intravenous glucose tolerance test, hepatic lipid content was assessed by 1 H magnetic resonance spectroscopy, serum interleukin (IL)-6 and IL-18 were assessed by ELISA, and peripheral and autonomic neuropathy were assessed by functional and clinical measures. Participants were followed up for up to 16 years. We also investigated heart failure and all-cause mortality in 794 individuals with type 2 diabetes undergoing invasive coronary diagnostics from the Ludwigshafen Risk and Cardiovascular Health (LURIC) cohort. FINDINGS: There were gradients of clamp-measured insulin sensitivity (both dimensions: p<0·0001) and insulin secretion (pdim1<0·0001, pdim2=0·00097) across the tree. Individuals in the region with the lowest insulin sensitivity had the highest hepatic lipid content (n=205, pdim1<0·0001, pdim2=0·037), pro-inflammatory biomarkers (IL-6: n=348, pdim1<0·0001, pdim2=0·013; IL-18: n=350, pdim1<0·0001, pdim2=0·38), and elevated cardiovascular risk (nevents=143, pdim1=0·14, pdim2<0·00081), whereas individuals positioned in the branch with the lowest insulin secretion were more prone to require insulin therapy (nevents=85, pdim1=0·032, pdim2=0·12) and had the highest risk of diabetic sensorimotor polyneuropathy (nevents=184, pdim1=0·012, pdim2=0·044) and cardiac autonomic neuropathy (nevents=118, pdim1=0·0094, pdim2=0·06). In the LURIC cohort, all-cause mortality was highest in the tree branch showing insulin resistance (nevents=488, pdim1=0·12, pdim2=0·0032). Significant gradients differentiated individuals having heart failure with preserved ejection fraction from those who had heart failure with reduced ejection fraction. INTERPRETATION: These data define the pathophysiological underpinnings of the tree structure, which has the potential to stratify diabetes-related complications on the basis of routinely available variables and thereby expand the toolbox of precision diabetes diagnosis. FUNDING: German Diabetes Center, German Federal Ministry of Health, Ministry of Culture and Science of the state of North Rhine-Westphalia, German Federal Ministry of Education and Research, German Diabetes Association, German Center for Diabetes Research, European Community, German Research Foundation, and Schmutzler Stiftung.

Brain insulin action on peripheral insulin sensitivity in women depends on menstrual cycle phase.

Insulin action in the human brain modulates eating behaviour, whole-body metabolism and body fat distribution1,2. In particular, brain insulin action increases whole-body insulin sensitivity, but these studies were mainly performed in lean men3,4. Here we investigate metabolic and hypothalamic effects of brain insulin action in women with a focus on the impact of menstrual cycle ( ClinicalTrials.gov registration: NCT03929419 ).Eleven women underwent four hyperinsulinemic-euglycemic clamps, two in the follicular phase and two in the luteal phase. Brain insulin action was introduced using nasal insulin spray5-7 and compared to placebo spray in a fourfold crossover design with change in glucose infusion rate as the primary endpoint. Here we show that during the follicular phase, more glucose has to be infused after administration of nasal insulin than after administration of placebo. This remains significant after adjustment for blood glucose and insulin. During the luteal phase, no significant influence of brain insulin action on glucose infusion rate is detected after adjustment for blood glucose and insulin (secondary endpoint). In 15 other women, hypothalamic insulin sensitivity was assessed in a within-subject design by functional magnetic resonance imaging with intranasal insulin administration8. Hypothalamus responsivity is influenced by insulin in the follicular phase but not the luteal phase.Our study therefore highlights that brain insulin action improves peripheral insulin sensitivity also in women but only during the follicular phase. Thus, brain insulin resistance could contribute to whole-body insulin resistance in the luteal phase of the menstrual cycle.

Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis.

Impaired proinsulin-to-insulin processing in pancreatic ß-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 ß-cell function and demise is unclear. Here we define the lipid signature of T2D-associated ß-cell failure, including an imbalance of specific very-long-chain SLs and long-chain SLs. ß-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 ß-cell function and T2D-associated ß-cell failure.

Serum neurofilament light chain: a novel biomarker for early diabetic sensorimotor polyneuropathy.

AIMS/HYPOTHESIS: No established blood-based biomarker exists to monitor diabetic sensorimotor polyneuropathy (DSPN) and evaluate treatment response. The neurofilament light chain (NFL), a blood biomarker of neuroaxonal damage in several neurodegenerative diseases, represents a potential biomarker for DSPN. We hypothesised that higher serum NFL levels are associated with prevalent DSPN and nerve dysfunction in individuals recently diagnosed with diabetes. METHODS: This cross-sectional study included 423 adults with type 1 and type 2 diabetes and known diabetes duration of less than 1 year from the prospective observational German Diabetes Study cohort. NFL was measured in serum samples of fasting participants in a multiplex approach using proximity extension assay technology. DSPN was assessed by neurological examination, nerve conduction studies and quantitative sensory testing. Associations of serum NFL with DSPN (defined according to the Toronto Consensus criteria) were estimated using Poisson regression, while multivariable linear and quantile regression models were used to assess associations with nerve function measures. In exploratory analyses, other biomarkers in the multiplex panel were also analysed similarly to NFL. RESULTS: DSPN was found in 16% of the study sample. Serum NFL levels increased with age. After adjustment for age, sex, waist circumference, height, HbA1c, known diabetes duration, diabetes type, cholesterol, eGFR, hypertension, CVD, use of lipid-lowering drugs and use of non-steroidal anti-inflammatory drugs, higher serum NFL levels were associated with DSPN (RR [95% CI] per 1-normalised protein expression increase, 1.92 [1.50, 2.45], p<0.0001), slower motor (all p<0.0001) and sensory (all p≤0.03) nerve conduction velocities, lower sural sensory nerve action potential (p=0.0004) and higher thermal detection threshold to warm stimuli (p=0.023 and p=0.004 for hand and foot, respectively). There was no evidence for associations between other neurological biomarkers and DSPN or nerve function measures. CONCLUSIONS/INTERPRETATION: Our findings in individuals recently diagnosed with diabetes provide new evidence associating higher serum NFL levels with DSPN and peripheral nerve dysfunction. The present study advocates NFL as a potential biomarker for DSPN.

Dietary lipid droplet structure in postnatal life improves hepatic energy and lipid metabolism in a mouse model for postnatal programming.

Early-life diets may have a long-lasting impact on metabolic health. This study tested the hypothesis that an early-life diet with large, phospholipid-coated lipid droplets (Concept) induces sustained improvements of hepatic mitochondrial function and metabolism. Young C57BL/6j mice were fed Concept or control (CTRL) diet from postnatal day 15 (PN15) to PN42, followed by western style (WSD) or standard rodent diet (AIN) until PN98. Measurements comprised body composition, insulin resistance (HOMA-IR), tricarboxylic acid (TCA) cycle- and ß-oxidation-related hepatic oxidative capacity using high-resolution respirometry, mitochondrial dynamics, mediators of insulin resistance (diacylglycerols, DAG) or ceramides) in subcellular compartments as well as systemic oxidative stress. Concept feeding increased TCA cycle-related respiration by 33% and mitochondrial fusion protein-1 by 65% at PN42 (both p 0.05). At PN98, CTRL, but not Concept, mice developed hyperinsulinemia (CTRL/AIN 0.22 ± 0.44 vs. CTRL/WSD 1.49 ± 0.53 nmol/l, p 0.05 and Concept/AIN 0.20 ± 0.38 vs. Concept/WSD 1.00 ± 0.29 nmol/l, n.s.) and insulin resistance after WSD (CTRL/AIN 107 ± 23 vs. CTRL/WSD 738 ± 284, p 0.05 and Concept/AIN 109 ± 24 vs. Concept/WSD 524 ± 157, n.s.). WSD-induced liver weight was 18% lower in adult Concept-fed mice and ß-oxidation-related respiration was 69% higher (p 0.05; Concept/WSD vs. Concept/AIN) along with lower plasma lipid peroxides (CTRL/AIN 4.85 ± 0.28 vs. CTRL/WSD 5.73 ± 0.47 µmol/l, p 0.05 and Concept/AIN 4.49 ± 0.31 vs. Concept/WSD 4.42 ± 0.33 µmol/l, n.s.) and were in part protected from WSD-induced increase in hepatic cytosolic DAG C16:0/C18:1. Early-life feeding of Concept partly protected from WSD-induced insulin resistance and systemic oxidative stress, potentially via changes in specific DAG and mitochondrial function, highlighting the role of early life diets on metabolic health later in life.

Leukocyte Counts and T-Cell Frequencies Differ Between Novel Subgroups of Diabetes and Are Associated With Metabolic Parameters and Biomarkers of Inflammation.

Frequencies of circulating immune cells are altered in those with type 1 and type 2 diabetes compared with healthy individuals and are associated with insulin sensitivity, glycemic control, and lipid levels. This study aimed to determine whether specific immune cell types are associated with novel diabetes subgroups. We analyzed automated white blood cell counts (n = 669) and flow cytometric data (n = 201) of participants in the German Diabetes Study with recent-onset (<1 year) diabetes, who were allocated to five subgroups based on data-driven analysis of clinical variables. Leukocyte numbers were highest in severe insulin-resistant diabetes (SIRD) and mild obesity-related diabetes (MOD) and lowest in severe autoimmune diabetes (SAID). CD4+ T-cell frequencies were higher in SIRD versus SAID, MOD, and mild age-related diabetes (MARD), and frequencies of CCR4+ regulatory T cells were higher in SIRD versus SAID and MOD and in MARD versus SAID. Pairwise differences between subgroups were partially explained by differences in clustering variables. Frequencies of CD4+ T cells were positively associated with age, BMI, HOMA2 estimate of ß-cell function (HOMA2-B), and HOMA2 estimate of insulin resistance (HOMA2-IR), and frequencies of CCR4+ regulatory T cells with age, HOMA2-B, and HOMA2-IR. In conclusion, different leukocyte profiles exist between novel diabetes subgroups and suggest distinct inflammatory processes in these diabetes subgroups.

Differences in Biomarkers of Inflammation Between Novel Subgroups of Recent-Onset Diabetes.

A novel clustering approach identified five subgroups of diabetes with distinct progression trajectories of complications. We hypothesized that these subgroups differ in multiple biomarkers of inflammation. Serum levels of 74 biomarkers of inflammation were measured in 414 individuals with recent adult-onset diabetes from the German Diabetes Study (GDS) allocated to five subgroups based on data-driven cluster analysis. Pairwise differences between subgroups for biomarkers were assessed with generalized linear mixed models before (model 1) and after (model 2) adjustment for the clustering variables. Participants were assigned to five subgroups: severe autoimmune diabetes (21%), severe insulin-deficient diabetes (SIDD) (3%), severe insulin-resistant diabetes (SIRD) (9%), mild obesity-related diabetes (32%), and mild age-related diabetes (35%). In model 1, 23 biomarkers showed one or more pairwise differences between subgroups (Bonferroni-corrected P < 0.0007). Biomarker levels were generally highest in SIRD and lowest in SIDD. All 23 biomarkers correlated with one or more of the clustering variables. In model 2, three biomarkers (CASP-8, EN-RAGE, IL-6) showed at least one pairwise difference between subgroups (e.g., lower CASP8, EN-RAGE, and IL-6 in SIDD vs. all other subgroups, all P < 0.0007). Thus, novel diabetes subgroups show multiple differences in biomarkers of inflammation, underlining a prominent role of inflammatory pathways in particular in SIRD.

Vitamin B12 and Folate Concentrations in Recent-onset Type 2 Diabetes and the Effect of Metformin Treatment.

CONTEXT: Vitamin B12 and folate deficiency are not only linked to hematological, neurological, and cardiovascular diseases, but are also associated with insulin resistance. Metformin can decrease vitamin B12 and folate concentrations. OBJECTIVE: To examine (1) effects of short-term metformin treatment on serum holotranscobalamin (holoTC) and folate and (2) their association with insulin sensitivity in recent-onset type 2 diabetes. DESIGN: This cross-sectional analysis comprised patients (known disease duration <12 months) on metformin monotherapy (MET, n = 123, 81 males, 53 ±â€…12 years) or nonpharmacological treatment (NPT, n = 126, 77 males, 54 ±â€…11 years) of the German Diabetes Study. MAIN OUTCOME MEASURES: HoloTC (enzyme-linked immunosorbent assay), cobalamin, and folate (electrochemiluminescence); beta-cell function and whole-body insulin sensitivity, measured during fasting (HOMA-B, HOMA-IR) and intravenous glucose tolerance tests combined with hyperinsulinemic-euglycemic clamp tests. RESULTS: HoloTC (105.4 [82.4, 128.3] vs 97 [79.7, 121.9] pmol/L) and folate concentrations (13.4 [9.3, 19.3] vs 12.7 [9.3, 22.0] nmol/L) were similar in both groups. Overall, holoTC was not associated with fasting or glucose-stimulated beta-cell function and insulin-stimulated glucose disposal. Cobalamin measurements yielded similar results in representative subgroups. In NPT but not MET, folate levels were inversely correlated with HOMA-IR (r = -0.239, P = .007). Folate levels did not relate to insulin sensitivity or insulin secretion in the whole cohort and in each group separately after adjustment for age, body mass index, and sex. CONCLUSIONS: Metformin does not affect circulating holoTC and folate concentrations in recent-onset type 2 diabetes, rendering monitoring of vitamin B12 and folate dispensable, at least during the first 6 months after diagnosis or initiation of metformin.

Metabolic profiling of dormant Mycolicibacterium smegmatis cells' reactivation reveals a gradual assembly of metabolic processes.

INTRODUCTION: Under gradual acidification of the culture medium mycobacterial cells transit into a specific state characterized by low level of metabolic activity and morphological alterations. This state of non-replicative persistence (dormancy) is directly linked to physiological drug resistance, which complicates the efforts to eradicate the latent forms of TB. In order to find new anti-latent TB compounds, the metabolic processes which may occur in the state of dormancy and during the transition into the active state (reactivation) should be characterized. OBJECTIVES: In the current study we analyzed the untargeted metabolomic profiles of dormant and reactivating Mycolicibacterium smegmatis cells (a model microorganism, bearing many common physiological traits of MTB), on the global scale level, since the characterization and analysis of the metabolites' dynamics would provide a comprehensive overview on global biochemical responses of the bacteria to stress conditions. METHODS: The reactivation process was tracked by measuring the value of membrane potential, applying a ratio-metric approach, by the method of flow-cytometry. The crucial timepoints were selected and the bacteria were sampled to LC-MS metabolic profiling. RESULTS: Reactivation of these cells after 60 days of storage revealed that this process proceeds in two stages: (I) a period, which lasts for 10 h and is characterized by a constant CFU number, unchangeable cell size, a minuscule increase of respiratory activity and a noticeable increase in membrane potential value, indicating the onset of the first metabolic processes during this time interval; the second phase (10-26 h) is characterized by acceleration of endogenous respiration, changes in the size of the cells and it finishes with the beginning of cells division. Analysis of the changes in the relative abundances of KEGG-annotated metabolites revealed that a significant number of metabolites, such as stearic acid, glycerol, D-glucose, trehalose-6-phosphate decrease their concentrations over the reactivation time, whereas in contrast, such metabolites as dodecanoic acid, mycobactin S, and other compounds of PG/AG biosynthesis are synthesized during reactivation. Differential analysis of metabolic profiles disclosed the activation of a number of metabolic pathways at the early reactivation stage: biosynthesis of secondary metabolites, purine and pyrimidine metabolism, glycerophospholipid and fatty acids metabolism etc. CONCLUSION: The data obtained indicate, despite the long-term storage of dormant cells in a state of minimal metabolic activity, according to metabolic profiling, they still retained a large number of metabolites. In the process of reactivation, the incremental stochastic assembly of the complete metabolic pathways occurs.

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.

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 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 down-regulated in both young and mature leaves at both time points. Combined analyses of gene coexpression 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 cross talk between FL acclimation and systemic acquired resistance-like gene expression in young leaves.

Characterization and engineering of a carotenoid biosynthesis operon from Bacillus megaterium.

Bacillus megaterium belongs to the group of pigmented bacilli producing carotenoids that ensure self-protection from UV radiation-induced and collateral oxidative damage. Metabolite profiling of strain MS941 revealed the presence of the C30 carotenoids 4,4'-diapophytofluene and 4,4'-diaponeurosporenic acid. A gene function analysis demonstrated the presence of a corresponding C30 carotenoid biosynthetic pathway with pharmaceutical importance. We identified a gene cluster comprising putative genes for a farnesyl diphosphate synthase (IspA), a diapophytoene synthase (CrtM) and three distinct diapophytoene desaturases (CrtN1-3). Intriguingly, crtM was organized in an operon together with two of the identified crtN genes. The individual activities of the encoded enzymes were determined by heterologous expression and product analysis in the non-carotenogenic model organism Escherichia coli. Our experimental data show that the first catalytic steps of C30 carotenoid biosynthesis in B. megaterium share significant similarity to the corresponding biosynthetic pathway of Staphylococcus aureus. The biosynthesis of farnesyl diphosphates and their subsequent condensation to form 4,4'-diapophytoene are catalyzed by the identified IspA and CrtM, respectively. The following desaturation reactions to form 4,4'-diaponeurosporene, however, require the activities of multiple diapophytoene desaturases. A biosynthetic operon was engineered and successfully expressed in an E. coli whole-cell system creating a cell factory for a high-yield production of the C30 carotenoid 4,4'-diaponeurosporene which has promising potential in the treatment of various inflammatory diseases.

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.

The Mycobacterium avium ssp. paratuberculosis specific mptD gene is required for maintenance of the metabolic homeostasis necessary for full virulence in mouse infections.

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease, a chronic granulomatous enteritis in ruminants. Furthermore, infections of humans with MAP have been reported and a possible association with Crohn's disease and diabetes type I is currently discussed. MAP owns large sequence polymorphisms (LSPs) that were exclusively found in this mycobacteria species. The relevance of these LSPs in the pathobiology of MAP is still unclear. The mptD gene (MAP3733c) of MAP belongs to a small group of functionally uncharacterized genes, which are not present in any other sequenced mycobacteria species. mptD is part of a predicted operon (mptABCDEF), encoding a putative ATP binding cassette-transporter, located on the MAP-specific LSP14. In the present study, we generated an mptD knockout strain (MAPΔmptD) by specialized transduction. In order to investigate the potential role of mptD in the host, we performed infection experiments with macrophages. By this, we observed a significantly reduced cell number of MAPΔmptD early after infection, indicating that the mutant was hampered with respect to adaptation to the early macrophage environment. This important role of mptD was supported in mouse infection experiments where MAPΔmptD was significantly attenuated after peritoneal challenge. Metabolic profiling was performed to determine the cause for the reduced virulence and identified profound metabolic disorders especially in the lipid metabolism of MAPΔmptD. Overall our data revealed the mptD gene to be an important factor for the metabolic adaptation of MAP required for persistence in the host.

The plant growth promoting substance, lumichrome, mimics starch, and ethylene-associated symbiotic responses in lotus and tomato roots.

Symbiosis involves responses that maintain the plant host and symbiotic partner's genetic program; yet these cues are far from elucidated. Here we describe the effects of lumichrome, a flavin identified from Rhizobium spp., applied to lotus (Lotus japonicus) and tomato (Solanum lycopersicum). Combined transcriptional and metabolite analyses suggest that both species shared common pathways that were altered in response to this application under replete, sterile conditions. These included genes involved in symbiosis, as well as transcriptional and metabolic responses related to enhanced starch accumulation and altered ethylene metabolism. Lumichrome priming also resulted in altered colonization with either Mesorhizobium loti (for lotus) or Glomus intraradices/G. mossea (for tomato). It enhanced nodule number but not nodule formation in lotus; while leading to enhanced hyphae initiation and delayed arbuscule maturation in tomato.

Network analysis of enzyme activities and metabolite levels and their relationship to biomass in a large panel of Arabidopsis accessions.

Natural genetic diversity provides a powerful resource to investigate how networks respond to multiple simultaneous changes. In this work, we profile maximum catalytic activities of 37 enzymes from central metabolism and generate a matrix to investigate species-wide connectivity between metabolites, enzymes, and biomass. Most enzyme activities change in a highly coordinated manner, especially those in the Calvin-Benson cycle. Metabolites show coordinated changes in defined sectors of metabolism. Little connectivity was observed between maximum enzyme activities and metabolites, even after applying multivariate analysis methods. Measurements of posttranscriptional regulation will be required to relate these two functional levels. Individual enzyme activities correlate only weakly with biomass. However, when they are used to estimate protein abundances, and the latter are summed and expressed as a fraction of total protein, a significant positive correlation to biomass is observed. The correlation is additive to that obtained between starch and biomass. Thus, biomass is predicted by two independent integrative metabolic biomarkers: preferential investment in photosynthetic machinery and optimization of carbon use.

C acid decarboxylases required for C photosynthesis are active in the mid-vein of the C species Arabidopsis thaliana, and are important in sugar and amino acid metabolism.

Cells associated with veins of petioles of C(3) tobacco possess high activities of the decarboxylase enzymes required in C(4) photosynthesis. It is not clear whether this is the case in other C(3) species, nor whether these enzymes provide precursors for specific biosynthetic pathways. Here, we investigate the activity of C(4) acid decarboxylases in the mid-vein of Arabidopsis, identify regulatory regions sufficient for this activity, and determine the impact of removing individual isoforms of each protein on mid-vein metabolite profiles. This showed that radiolabelled malate and bicarbonate fed to the xylem stream were incorporated into soluble and insoluble material in the mid-vein of Arabidopsis leaves. Compared with the leaf lamina, mid-veins possessed high activities of NADP-dependent malic enzyme (NADP-ME), NAD-dependent malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PEPCK). Transcripts derived from both NAD-ME, one PCK and two of the four NADP-ME genes were detectable in these veinal cells. The promoters of each decarboxylase gene were sufficient for expression in mid-veins. Analysis of insertional mutants revealed that cytosolic NADP-ME2 is responsible for 80% of NADP-ME activity in mid-veins. Removing individual decarboxylases affected the abundance of amino acids derived from pyruvate and phosphoenolpyruvate. Reducing cytosolic NADP-ME activity preferentially affected the sugar content, whereas abolishing NAD-ME affected both the amino acid and the glucosamine content of mid-veins.