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Type 2 diabetes is a challenge in modern healthcare, and animal models are necessary to identify underlying mechanisms, where we can achieve much better environmental control than what is practical in human studies. The Nile rat (Arvicanthis niloticus) develops diet-induced diabetes rapidly on a conventional rodent chow diet without genetic or chemical manipulation. Unlike common laboratory models, the outbred Nile rat model is diurnal and can progress to advanced diabetic complications, better mimicking the human condition. Some human studies indicate that compared to fasting glucose, post-prandial blood glucose is more sensitive to the initial stages of diabetes, suggesting that we should capture the non-fasted state to study early diabetes. However, it is unknown if ad libitum feeding in the Nile rats leads to increased variance thus masking diabetes-related metabolic changes in the plasma. In this study, we compared the repeatability within triplicate non-fasted or fasted plasma samples and assessed prediction of impaired glucose tolerance in fasted and non-fasted plasma. We used liquid chromatography-mass spectrometry lipidomics and polar metabolomics to measure relative metabolite abundances in the plasma samples. Metabolite measurements in non-fasted plasma were less variable than measurements in fasted plasma. We detected 66 metabolites in non-fasted plasma associated with glucose tolerance in elastic net and individual metabolite linear regression models. Low metabolite replicate variance was reproduced in a cohort of mature 30-week male and female Nile rats. Our results support using non-fasted plasma metabolomics for early detection of impaired glucose tolerance in Nile rats.

The oxidant hydrogen peroxide serves as a signaling molecule that alters many aspects of cardiovascular functions. Recent studies suggest that cytoglobin – a hemoglobin expressed in the vasculature – may promote electron transfer reactions with proposed functions in hydrogen peroxide decomposition. Here, we determined the extent to which cytoglobin regulates intracellular hydrogen peroxide and established mechanisms. We found that cytoglobin decreased the hyperoxidation of peroxiredoxins and maintained the activity of peroxiredoxin 2 following challenge with exogenous hydrogen peroxide. Cytoglobin promoted a reduced intracellular environment and facilitated the reduction of the thiol-based hydrogen peroxide sensor Hyper7 after bolus addition of hydrogen peroxide. Cytoglobin also limited the inhibitory effect of hydrogen peroxide on glycolysis and reversed the oxidative inactivation of the glycolytic enzyme GAPDH. Our results indicate that cytoglobin in cells exists primarily as oxyferrous cytoglobin (CygbFe2+-O2) with its cysteine residues in the reduced form. We found that the specific substitution of one of two cysteine residues on cytoglobin (C83A) inhibited the reductive activity of cytoglobin on Hyper7 and GAPDH. Carotid arteries from cytoglobin knockout mice were more sensitive to glycolytic inhibition by hydrogen peroxide than arteries from wildtype mice. Together, these results support a role for cytoglobin in regulating intracellular redox signals associated with hydrogen peroxide through oxidation of its cysteine residues, independent of hydrogen peroxide reaction at its heme center.

Multi-omics analysis is a powerful and increasingly utilized approach to gain insight into complex biological systems. One major hindrance with multi-omics, however, is the lengthy and wasteful sample preparation process. Preparing samples for mass spectrometry (MS)-based multi-omics involves extraction of metabolites and lipids with organic solvents, precipitation of proteins, and overnight digestion of proteins. These existing workflows are disparate and laborious. Here, we present a simple, efficient, and unified approach to prepare lipids, metabolites, and proteins for MS analysis. Our approach, termed the Bead-enabled Accelerated Monophasic Multi-omics (BAMM) method, combines an

Organisms have evolved elaborate physiological pathways that regulate growth, proliferation, metabolism, and stress response. These pathways must be properly coordinated to elicit the appropriate response to an ever-changing environment. While individual pathways have been well studied in a variety of model systems, there remains much to uncover about how pathways are integrated to produce systemic changes in a cell, especially in dynamic conditions. We previously showed that deletion of Protein Kinase A (PKA) regulatory subunitBCY1can decouple growth and metabolism inSaccharomyces cerevisiaeengineered for anaerobic xylose fermentation, allowing for robust fermentation in the absence of division. This provides an opportunity to understand how PKA signaling normally coordinates these processes. Here, we integrated transcriptomic, lipidomic, and phosphor-proteomic responses upon a glucose to xylose shift across a series of strains with different genetic mutations promoting either coupled or decoupled xylose-dependent growth and metabolism. Together, results suggested that defects in lipid homeostasis limit growth in thebcy1Δstrain despite robust metabolism. To further understand this mechanism, we performed adaptive laboratory evolutions to re-evolve coupled growth and metabolism in thebcy1Δparental strain. Genetic mutations in PKA subunitTPK1and lipid regulatorOPI1, among other genes underscored a role for lipid homeostasis, which was further supported by evolved changes in lipid profiles and gene expression. We suggest several models for how cells coordinate growth, metabolism, and other responses in budding yeast and how restructuring these processes enables anaerobic xylose utilization.Author SummaryAll organisms utilize an energy source to generate the cellular resources needed to grow and divide. These individual processes have been well study, but the coordination and crosstalk between the process is not well understood. To study growth and metabolism coupling, we used a yeast strain that was genetically engineered to ferment the sugar xylose but lacked growth on the sugar. The decoupled growth and metabolism was caused by a single gene deletion in a highly conserved signaling pathway found in all eukaryotes. While our work is focused on xylose metabolism, we address the fundamental question of how cells coordinate growth with metabolism under non-ideal conditions. We identified vast changes in gene expression that implicated altered regulatory mechanisms involved in lipid metabolism correlating with decouple growth and metabolism. Our work highlights the complexity of engineering new cellular functions and that global regulatory modifications, rather than altering individual pathways, may be required for broad cellular changes.

Background Developing economically viable pathways to produce renewable energy has become an important research theme in recent years. Lignocellulosic biomass is a promising feedstock that can be converted into second-generation biofuels and bioproducts. Global warming has adversely affected climate change causing many environmental changes that have impacted earth surface temperature and rainfall patterns. Recent research has shown that environmental growth conditions altered the composition of drought-stressed switchgrass and directly influenced the extent of biomass conversion to fuels by completely inhibiting yeast growth during fermentation. Our goal in this project was to find a way to overcome the microbial inhibition and characterize specific compounds that led to this inhibition. Additionally, we also determined if these microbial inhibitors were plant-generated compounds, by-products of the pretreatment process, or a combination of both. Results Switchgrass harvested in drought (2012) and non-drought (2010) years were pretreated using Ammonia Fiber Expansion (AFEX). Untreated and AFEX processed samples were then extracted using solvents (i.e., water, ethanol, and ethyl acetate) to selectively remove potential inhibitory compounds and determine whether pretreatment affects the inhibition. High solids loading enzymatic hydrolysis was performed on all samples, followed by fermentation using engineered Saccharomyces cerevisiae. Fermentation rate, cell growth, sugar consumption, and ethanol production were used to evaluate fermentation performance. We found that water extraction of drought-year switchgrass before AFEX pretreatment reduced the inhibition of yeast fermentation. The extracts were analyzed using liquid chromatography–mass spectrometry (LC–MS) to detect compounds enriched in the extracted fractions. Saponins, a class of plant-generated triterpene or steroidal glycosides, were found to be significantly more abundant in the water extracts from drought-year (inhibitory) switchgrass. The inhibitory nature of the saponins in switchgrass hydrolysate was validated by spiking commercially available saponin standard (protodioscin) in non-inhibitory switchgrass hydrolysate harvested in normal year. Conclusions Adding a water extraction step prior to AFEX-pretreatment of drought-stressed switchgrass effectively overcame inhibition of yeast growth during bioethanol production. Saponins appear to be generated by the plant as a response to drought as they were significantly more abundant in the drought-stressed switchgrass water extracts and may contribute toward yeast inhibition in drought-stressed switchgrass hydrolysates.