Reconstruction of a genome-scale metabolic model and in-silico flux analysis of Aspergillus tubingensis: a non-mycotoxinogenic citric acid-producing fungus.

BACKGROUND: Aspergillus tubingensis is a citric acid-producing fungus that can utilize sugars in hydrolysate of lignocellulosic biomass such as sugarcane bagasse and, unlike A. niger, does not produce mycotoxins. To date, no attempt has been made to model its metabolism at genome scale. RESULTS: Here, we utilized the whole-genome sequence (34.96 Mb length) and the measured biomass composition to reconstruct a genome-scale metabolic model (GSMM) of A. tubingensis DJU120 strain. The model, named iMK1652, consists of 1652 genes, 1657 metabolites and 2039 reactions distributed over four cellular compartments. The model has been extensively curated manually. This included removal of dead-end metabolites and generic reactions, addition of secondary metabolite pathways and several transporters. Several mycotoxin synthesis pathways were either absent or incomplete in the genome, providing a genomic basis for the non-toxinogenic nature of this species. The model was further refined based on the experimental phenotypic microarray (Biolog) data. The model closely captured DJU120 fermentative data on glucose, xylose, and phosphate consumption, as well as citric acid and biomass production, showing its applicability to capture citric acid fermentation of lignocellulosic biomass hydrolysate. CONCLUSIONS: The model offers a framework to conduct metabolic systems biology investigations and can act as a scaffold for integrative modelling of A. tubingensis.

Marine cyanobacterial biomass is an efficient feedstock for fungal bioprocesses.

BACKGROUND: Marine cyanobacteria offer many sustainability advantages, such as the ability to fix atmospheric CO2, very fast growth and no dependence on freshwater for culture. Cyanobacterial biomass is a rich source of sugars and proteins, two essential nutrients for culturing any heterotroph. However, no previous study has evaluated their application as a feedstock for fungal bioprocesses. RESULTS: In this work, we cultured the marine cyanobacterium Synechococcus sp. PCC 7002 in a 3-L externally illuminated bioreactor with working volume of 2 L with a biomass productivity of ~ 0.8 g L-1 day-1. Hydrolysis of the biomass with acids released proteins and hydrolyzed glycogen while hydrolysis of the biomass with base released only proteins but did not hydrolyze glycogen. Among the different acids tested, treatment with HNO3 led to the highest release of proteins and glucose. Cyanobacterial biomass hydrolysate (CBH) prepared in HNO3 was used as a medium to produce cellulase enzyme by the Penicillium funiculosum OAO3 strain while CBH prepared in HCl and treated with charcoal was used as a medium for citric acid by Aspergillus tubingensis. Approximately 50% higher titers of both products were obtained compared to traditional media. CONCLUSIONS: These results show that the hydrolysate of marine cyanobacteria is an effective source of nutrients/proteins for fungal bioprocesses.

Integration of Aspergillus niger transcriptomic profile with metabolic model identifies potential targets to optimise citric acid production from lignocellulosic hydrolysate.

BACKGROUND: Citric acid is typically produced industrially by Aspergillus niger-mediated fermentation of a sucrose-based feedstock, such as molasses. The fungus Aspergillus niger has the potential to utilise lignocellulosic biomass, such as bagasse, for industrial-scale citric acid production, but realising this potential requires strain optimisation. Systems biology can accelerate strain engineering by systematic target identification, facilitated by methods for the integration of omics data into a high-quality metabolic model. In this work, we perform transcriptomic analysis to determine the temporal expression changes during fermentation of bagasse hydrolysate and develop an evolutionary algorithm to integrate the transcriptomic data with the available metabolic model to identify potential targets for strain engineering. RESULTS: The novel integrated procedure matures our understanding of suboptimal citric acid production and reveals potential targets for strain engineering, including targets consistent with the literature such as the up-regulation of citrate export and pyruvate carboxylase as well as novel targets such as the down-regulation of inorganic diphosphatase. CONCLUSIONS: In this study, we demonstrate the production of citric acid from lignocellulosic hydrolysate and show how transcriptomic data across multiple timepoints can be coupled with evolutionary and metabolic modelling to identify potential targets for further engineering to maximise productivity from a chosen feedstock. The in silico strategies employed in this study can be applied to other biotechnological goals, assisting efforts to harness the potential of microorganisms for bio-based production of valuable chemicals.

Metabolic systems biology and multi-omics of cyanobacteria: Perspectives and future directions.

Cyanobacteria are oxygenic photoautotrophs whose metabolism contains key biochemical pathways to fix atmospheric CO2 and synthesize various metabolites. The development of bioengineering tools has enabled the manipulation of cyanobacterial chassis to produce various valuable bioproducts photosynthetically. However, effective utilization of cyanobacteria as photosynthetic cell factories needs a detailed understanding of their metabolism and its interaction with other cellular processes. Implementing systems and synthetic biology tools has generated a wealth of information on various metabolic pathways. However, to design effective engineering strategies for further improvement in growth, photosynthetic efficiency, and enhanced production of target biochemicals, in-depth knowledge of their carbon/nitrogen metabolism, pathway fluxe distribution, genetic regulation and integrative analyses are necessary. In this review, we discuss the recent advances in the development of genome-scale metabolic models (GSMMs), omics analyses (metabolomics, transcriptomics, proteomics, fluxomics), and integrative modeling approaches to showcase the current understanding of cyanobacterial metabolism.

Salt-stress adaptation of yeast as a simple method to improve high-gravity fermentation in an industrial medium.

While Saccharomyces cerevisiae is a popular organism to produce ethanol, its fermentation performance is affected at high sugar concentrations due to osmotic stress. We hypothesized that adaptation under ionic stress conditions will improve the fermentation performance at high sugar concentrations due to cross-stress adaptation. We, therefore, adapted a high-performance yeast strain, S. cerevisiae CEN.PK 122, to increasing salt concentrations in an industrial medium. Control cells were adapted in the medium without added salt. The cells adapted to 3.5% (w/v) salt concentration demonstrated a superior performance when fermenting 10-30% (w/v) glucose. When fermenting 30% (w/v) glucose, the ethanol yields of the adapted cells (0.49 ± 0.01 g g-1) were about 30% higher than the control cells (0.37 ± 0.01 g g-1) and are comparable with the best reported to date for any medium employed. Similar improvements were also observed when fermenting 10% (w/v) sucrose. However, little improvement in fermentation was observed at the higher temperature tested (40 °C), even though the growth of the adapted cells was greater when tested in YPD medium. The improvements in fermentation at 30 °C were primarily related to the faster growth of the adapted cells and not to an increase in specific intake rates. Additionally, a significantly reduced lag phase was also observed when fermenting 30% (w/v) glucose. Thus, our work shows the application of a simple strategy to significantly improve high-gravity fermentation (HGF) performance through adaptation. KEY POINTS: • Cell adapted on 3.5% NaCl made 28% more ethanol when fermenting 30% glucose. • The adapted cells had reduced lag phase, grew faster, and produced less glycerol. • The improvements were not related to increased specific rates of production.

The Effect of Promoter and RBS Combination on the Growth and Glycogen Productivity of Sodium-Dependent Bicarbonate Transporter (SbtA) Overexpressing Synechococcus sp. PCC 7002 Cells.

Sodium dependent bicarbonate transporter, SbtA is a high-affinity, inducible bicarbonate transporter in cyanobacterial cells. Our previous work has shown that overexpression of this transporter can significantly increase growth and glycogen accumulation in Synechococcus sp. PCC 7002 cells. In this work, we have tested the effect of two different RBS sequences (RBS1: GGAGGA and RBS2: AGGAGA) and three different promoters (PcpcB, PcpcB 560, and PrbcL 2) on the growth and glycogen production in SbtA-overexpressing Synechococcus sp. PCC 7002 cells. Our results show that PcpcB or PcpcB 560 were more effective than PrbcL 2 in increasing the growth and glycogen content. The choice of RBS sequence had relatively minor effect, though RBS2 was more effective than RBS1. The transformant E, with PcpcB 560 and RBS2, showed the highest growth. The biomass after 5 days of growth on air or 1% CO2 was increased by about 90% in the strain E compared to PCC 7002 cells. All transformants overexpressing SbtA had higher glycogen content. However, growing the cells with bubbling of 1% CO2 did not increase cellular glycogen content any further. The strain E had about 80% higher glycogen content compared to WT PCC 7002 cells. Therefore, the glycogen productivity of the strain E grown with air-bubbling was about 2.5-fold that of the WT PCC 7002 cells grown similarly. Additionally, some of the transformants had higher chlorophyll content while all the transformants had higher carotenoid content compared to the PCC 7002 cells, suggesting interaction between carbon transport and pigment levels. Thus, this work shows that the choice of photosynthetic promoters and RBSs sequences can impact growth and glycogen accumulation in SbtA-overexpressing cells.

New paradigm in diatom omics and genetic manipulation.

Diatoms are one of the most heterogeneous eukaryotic plankton known for regulating earth's biogeochemical cycles and maintaining the marine ecosystems ever since the late Eocene epoch. The advent of multidisciplinary omics approach has both epitomized and revolutionized the nature of their chimeric genetic toolkit, ecophysiology, and metabolic adaptability as well as their interaction with other communities. In addition, advanced functional annotation of transcriptomic and proteomic data using cutting edge bioinformatics tools together with high-resolution genome-scale mathematical modeling has effectively proven as the catapult in solving genetic bottlenecks in microbial as well as diatom exploration. In this review, a corroborative summation of the robust work done in manipulating, engineering, and sequencing of the diatom genomes besides underpinning the holistic application of omics in transcription and translation has been discussed in order to shrewd their multifarious novel potential in the field of biotechnology and provide an insight into their dynamic evolutionary relevance.

A Genome-Scale Metabolic Model of Thalassiosira pseudonana CCMP 1335 for a Systems-Level Understanding of Its Metabolism and Biotechnological Potential.

Thalassiosira pseudonana is a transformable and biotechnologically promising model diatom with an ability to synthesise nutraceuticals such as fucoxanthin and store a significant amount of polyglucans and lipids including omega-3 fatty acids. While it was the first diatom to be sequenced, a systems-level analysis of its metabolism has not been done yet. This work presents first comprehensive, compartmentalized, and functional genome-scale metabolic model of the marine diatom Thalassiosira pseudonana CCMP 1335, which we have termed iThaps987. The model includes 987 genes, 2477 reactions, and 2456 metabolites. Comparison with the model of another diatom Phaeodactylum tricornutum revealed presence of 183 unique enzymes (belonging primarily to amino acid, carbohydrate, and lipid metabolism) in iThaps987. Model simulations showed a typical C3-type photosynthetic carbon fixation and suggested a preference of violaxanthin-diadinoxanthin pathway over violaxanthin-neoxanthin pathway for the production of fucoxanthin. Linear electron flow was found be active and cyclic electron flow was inactive under normal phototrophic conditions (unlike green algae and plants), validating the model predictions with previous reports. Investigation of the model for the potential of Thalassiosira pseudonana CCMP 1335 to produce other industrially useful compounds suggest iso-butanol as a foreign compound that can be synthesized by a single-gene addition. This work provides novel insights about the metabolism and potential of the organism and will be helpful to further investigate its metabolism and devise metabolic engineering strategies for the production of various compounds.

Biochemical Characteristics and a Genome-Scale Metabolic Model of an Indian Euryhaline Cyanobacterium with High Polyglucan Content.

Marine cyanobacteria are promising microbes to capture and convert atmospheric CO2 and light into biomass and valuable industrial bio-products. Yet, reports on metabolic characteristics of non-model cyanobacteria are scarce. In this report, we show that an Indian euryhaline Synechococcus sp. BDU 130192 has biomass accumulation comparable to a model marine cyanobacterium and contains approximately double the amount of total carbohydrates, but significantly lower protein levels compared to Synechococcus sp. PCC 7002 cells. Based on its annotated chromosomal genome sequence, we present a genome scale metabolic model (GSMM) of this cyanobacterium, which we have named as iSyn706. The model includes 706 genes, 908 reactions, and 900 metabolites. The difference in the flux balance analysis (FBA) predicted flux distributions between Synechococcus sp. PCC 7002 and Synechococcus sp. BDU130192 strains mimicked the differences in their biomass compositions. Model-predicted oxygen evolution rate for Synechococcus sp. BDU130192 was found to be close to the experimentally-measured value. The model was analyzed to determine the potential of the strain for the production of various industrially-useful products without affecting growth significantly. This model will be helpful to researchers interested in understanding the metabolism as well as to design metabolic engineering strategies for the production of industrially-relevant compounds.

Overexpression of bicarbonate transporters in the marine cyanobacterium Synechococcus sp. PCC 7002 increases growth rate and glycogen accumulation.

BACKGROUND: Synechococcus sp. PCC 7002 is an attractive organism as a feedstock and for photoautotrophic production of biofuels and biochemicals due to its fast growth and ability to grow in marine/brackish medium. Previous studies suggest that the growth of this organism is limited by the HCO3 - transport across the cytoplasmic membrane. Tools for genetic engineering are well established for this cyanobacterium, which makes it possible to overexpress genes of interest. RESULTS: In this work, we overexpressed two different native Na+-dependent carbon transporters viz., SbtA and BicA in Synechococcus sp. PCC 7002 cells under the influence of a strong light-inducible promoter and a strong RBS sequence. The overexpression of these transporters enhanced biomass by about 50%, increased intracellular glycogen about 50%, and increased extracellular carbohydrate up to threefold. Importantly, the biomass and glycogen productivity of the transformants with air bubbling was even higher than that of WT cells with 1% CO2 bubbling. The overexpression of these transporters was associated with an increased carotenoid content without altering the chl a content. CONCLUSIONS: Our work shows the utility of increased carbon transport in improving the growth as well as product formation in a marine cyanobacterium and will serve to increase the utility of this organism as a potential cell factory.

Genome-scale metabolic reconstruction and metabolic versatility of an obligate methanotroph Methylococcus capsulatus str. Bath.

The increase in greenhouse gases with high global warming potential such as methane is a matter of concern and requires multifaceted efforts to reduce its emission and increase its mitigation from the environment. Microbes such as methanotrophs can assist in methane mitigation. To understand the metabolic capabilities of methanotrophs, a complete genome-scale metabolic model (GSMM) of an obligate methanotroph, Methylococcus capsulatus str. Bath was reconstructed. The model contains 535 genes, 899 reactions and 865 metabolites and is named iMC535. The predictive potential of the model was validated using previously-reported experimental data. The model predicted the Entner-Duodoroff pathway to be essential for the growth of this bacterium, whereas the Embden-Meyerhof-Parnas pathway was found non-essential. The performance of the model was simulated on various carbon and nitrogen sources and found that M. capsulatus can grow on amino acids. The analysis of network topology of the model identified that six amino acids were in the top-ranked metabolic hubs. Using flux balance analysis, 29% of the metabolic genes were predicted to be essential, and 76 double knockout combinations involving 92 unique genes were predicted to be lethal. In conclusion, we have reconstructed a GSMM of a methanotroph Methylococcus capsulatus str. Bath. This is the first high quality GSMM of a Methylococcus strain which can serve as an important resource for further strain-specific models of the Methylococcus genus, as well as identifying the biotechnological potential of M. capsulatus Bath.

Brown and Brite: The Fat Soldiers in the Anti-obesity Fight.

Brown adipose tissue (BAT) is proposed to maintain thermal homeostasis through dissipation of chemical energy as heat by the uncoupling proteins (UCPs) present in their mitochondria. The recent demonstration of the presence of BAT in humans has invigorated research in this area. The research has provided many new insights into the biology and functioning of this tissue and the biological implications of its altered activities. Another finding of interest is browning of white adipose tissue (WAT) resulting in what is known as beige/brite cells, which have increased mitochondrial proteins and UCPs. In general, it has been observed that the activation of BAT is associated with various physiological improvements such as a reduction in blood glucose levels increased resting energy expenditure and reduced weight. Given the similar physiological functions of BAT and beige/ brite cells and the higher mass of WAT compared to BAT, it is likely that increasing the brite/beige cells in WATs may also lead to greater metabolic benefits. However, development of treatments targeting brown fat or WAT browning would require not only a substantial understanding of the biology of these tissues but also the effect of altering their activity levels on whole body metabolism and physiology. In this review, we present evidence from recent literature on the substrates utilized by BAT, regulation of BAT activity and browning by circulating molecules. We also present dietary and pharmacological activators of brown and beige/brite adipose tissue and the effect of physical exercise on BAT activity and browning.

FluxPyt: a Python-based free and open-source software for 13C-metabolic flux analyses.

13C-Metabolic flux analysis (MFA) is a powerful approach to estimate intracellular reaction rates which could be used in strain analysis and design. Processing and analysis of labeling data for calculation of fluxes and associated statistics is an essential part of MFA. However, various software currently available for data analysis employ proprietary platforms and thus limit accessibility. We developed FluxPyt, a Python-based truly open-source software package for conducting stationary 13C-MFA data analysis. The software is based on the efficient elementary metabolite unit framework. The standard deviations in the calculated fluxes are estimated using the Monte-Carlo analysis. FluxPyt also automatically creates flux maps based on a template for visualization of the MFA results. The flux distributions calculated by FluxPyt for two separate models: a small tricarboxylic acid cycle model and a larger Corynebacterium glutamicum model, were found to be in good agreement with those calculated by a previously published software. FluxPyt was tested in Microsoft™ Windows 7 and 10, as well as in Linux Mint 18.2. The availability of a free and open 13C-MFA software that works in various operating systems will enable more researchers to perform 13C-MFA and to further modify and develop the package.

Model-assisted metabolic engineering of Escherichia coli for long chain alkane and alcohol production.

Biologically-derived hydrocarbons are considered to have great potential as next-generation biofuels owing to the similarity of their chemical properties to contemporary diesel and jet fuels. However, the low yield of these hydrocarbons in biotechnological production is a major obstacle for commercialization. Several genetic and process engineering approaches have been adopted to increase the yield of hydrocarbon, but a model driven approach has not been implemented so far. Here, we applied a constraint-based metabolic modeling approach in which a variable demand for alkane biosynthesis was imposed, and co-varying reactions were considered as potential targets for further engineering of an E. coli strain already expressing cyanobacterial enzymes towards higher chain alkane production. The reactions that co-varied with the imposed alkane production were found to be mainly associated with the pentose phosphate pathway (PPP) and the lower half of glycolysis. An optimal modeling solution was achieved by imposing increased flux through the reaction catalyzed by glucose-6-phosphate dehydrogenase (zwf) and iteratively removing 7 reactions from the network, leading to an alkane yield of 94.2% of the theoretical maximum conversion determined by in silico analysis at a given biomass rate. To validate the in silico findings, we first performed pathway optimization of the cyanobacterial enzymes in E. coli via different dosages of genes, promoting substrate channelling through protein fusion and inducing substantial equivalent protein expression, which led to a 36-fold increase in alka(e)ne production from 2.8 mg/L to 102 mg/L. Further, engineering of E. coli based on in silico findings, including biomass constraint, led to an increase in the alka(e)ne titer to 425 mg/L (major components being 249 mg/L pentadecane and 160 mg/L heptadecene), a 148.6-fold improvement over the initial strain, respectively; with a yield of 34.2% of the theoretical maximum. The impact of model-assisted engineering was also tested for the production of long chain fatty alcohol, another commercially important molecule sharing the same pathway while differing only at the terminal reaction, and a titer of 1506 mg/L was achieved with a yield of 86.4% of the theoretical maximum. Moreover, the model assisted engineered strains had produced 2.54 g/L and 12.5 g/L of long chain alkane and fatty alcohol, respectively, in the bioreactor under fed-batch cultivation condition. Our study demonstrated successful implementation of a combined in silico modeling approach along with the pathway and process optimization in achieving the highest reported titers of long chain hydrocarbons in E. coli.

Novel quantitative insights into carbon sources for synthesis of poly hydroxybutyrate in Synechocystis PCC 6803.

Many freshwater cyanobacteria accumulate polyhydroxybutyrate (PHB) under nitrogen or phosphorus deprivation. While prior literature has shed lights on transcriptomic and metabolomic changes in the model cyanobacterium Synechocystis PCC 6803 cells, the quantitative contributions of the newly fixed carbon following nitrogen deprivation or the externally added acetate to PHB synthesis are not clear. Similarly, it is not clear how photomixotrophy affects precursor contributions. In this study, we show that (i) the pre-growth mode (photoautotrophic or photomixotrophic), while significantly impacting glycogen levels, does not have any significant effect on PHB levels, (ii) the carbon fixed following nitrogen deprivation contributes 26% of C for PHB synthesis in photoautotrophically pre-grown cells and its contribution to the PHB synthesis goes down with the addition of acetate at the resuspension phase or with photomixotrophic pre-growth, (iii) the acetate added at the start of nitrogen deprivation, doubles the intracellular PHB levels and contributes 44-48% to PHB synthesis and this value is not greatly affected by how the cells were pre-grown. Indirectly, the labeling studies also show that the intracellular C recycling is the most important source of precursors for PHB synthesis, contributing about 74-87% of the C for PHB synthesis in the absence of acetate. The addition of acetate significantly reduces its contribution. In photoautotrophic pre-growth followed by acetate addition under nitrogen starvation, the contribution of intracellular C reduces to about 34%. Thus, our study provides several novel quantitative insights on how prior nutritional status affects the precursor contributions for PHB synthesis.

Draft Genome Sequence of an Indian Marine Cyanobacterial Strain with Fast Growth and High Polyglucan Content.

Marine cyanobacteria play an important role in global carbon cycling and are a potential source of polyglucans for biotechnological purposes. This report provides the draft sequence of an Indian marine cyanobacterium, Synechococcus elongatus BDU 130192, which shows fast growth and high polyglucan content. The genome sequence will help in understanding the unique properties of this organism.

A Genome Scale Model of Geobacillus thermoglucosidasius (C56-YS93) reveals its biotechnological potential on rice straw hydrolysate.

Rice straw is a major crop residue which is burnt in many countries, creating significant air pollution. Thus, alternative routes for disposal of rice straw are needed. Biotechnological treatment of rice straw hydrolysate has potential to convert this agriculture waste into valuable biofuel(s) and platform chemicals. Geobacillus thermoglucosidasius is a thermophile with properties specially suited for use as a biocatalyst in lignocellulosic bioprocesses, such as high optimal temperature and tolerance to high levels of ethanol. However, the capabilities of G. thermoglucosidasius to utilise sugars in rice straw hydrolysate for making bioethanol and other platform chemicals have not been fully explored. In this work, we have created a genome scale metabolic model (denoted iGT736) of the organism containing 736 gene products, 1159 reactions and 1163 metabolites. The model was validated both by purely theoretical approaches and by comparing the behaviour of the model to previously published experimental results. The model was then used to determine the yields of a variety of platform chemicals from glucose and xylose - two primary sugars in rice straw hydrolysate. A comparison with results from a model of Escherichia coli shows that G. thermoglucosidasius is capable of producing a wider range of products, and that for the products also produced by E. coli, the yields are comparable. We also discuss strategies to utilise arabinose, a minor component of rice straw hydrolysate, and propose additional reactions to lead to the synthesis of xylitol, not currently produced by G. thermoglucosidasius. Our results provide additional motivation for the current exploration of the industrial potential of G. thermoglucosidasius and we make our model publicly available to aid the development of metabolic engineering strategies for this organism.

An Ester of ß-Hydroxybutyrate Regulates Cholesterol Biosynthesis in Rats and a Cholesterol Biomarker in Humans.

In response to carbohydrate deprivation or prolonged fasting the ketone bodies, ß-hydroxybutyrate (ßHB) and acetoacetate (AcAc), are produced from the incomplete ß-oxidation of fatty acids in the liver. Neither ßHB nor AcAc are well utilized for synthesis of sterols or fatty acids in human or rat liver. To study the effects of ketones on cholesterol homeostasis a novel ßHB ester (KE) ((R)-3-hydroxybutyl (R)-3-hydroxybutyrate) was synthesized and given orally to rats and humans as a partial dietary carbohydrate replacement. Rats maintained on a diet containing 30-energy % as KE with a concomitant reduction in carbohydrate had lower plasma cholesterol and mevalonate (-40 and -27 %, respectively) and in the liver had lower levels of the mevalonate precursors acetoacetyl-CoA and HMG-CoA (-33 and -54 %) compared to controls. Whole liver and membrane LDL-R as well as SREBP-2 protein levels were higher (+24, +67, and +91 %, respectively). When formulated into a beverage for human consumption subjects consuming a KE drink (30-energy %) had elevated plasma ßHB which correlated with decreased mevalonate, a liver cholesterol synthesis biomarker. Partial replacement of dietary carbohydrate with KE induced ketosis and altered cholesterol homeostasis in rats. In healthy individuals an elevated plasma ßHB correlated with lower plasma mevalonate.

A ketogenic diet increases brown adipose tissue mitochondrial proteins and UCP1 levels in mice.

We evaluated the effects of feeding a ketogenic diet (KD) for a month on general physiology with emphasis on brown adipose tissue (BAT) in mice. KD did not reduce the caloric intake, or weight or lipid content of BAT. Relative epididymal fat pads were 40% greater in the mice fed the KD (P = 0.06) while leptin was lower (P < 0.05). Blood glucose levels were 30% lower while D-ß-hydroxybutyrate levels were about 3.5-fold higher in the KD group. Plasma insulin and leptin levels in the KD group were about half of that of the mice fed NIH-31 pellets (chow group). Median mitochondrial size in the interscapular BAT (IBAT) of the KD group was about 60% greater, whereas the median lipid droplet size was about half of that in the chow group. Mitochondrial oxidative phosphorylation proteins were increased (1.5-3-fold) and the uncoupling protein 1 levels were increased by threefold in mice fed the KD. The levels of PPARγ, PGC-1α, and Sirt1 in KD group were 1.5-3-fold while level of Sirt3 was about half of that in the chow-fed group. IBAT cyclic AMP levels were 60% higher in the KD group and cAMP response element binding protein was 2.5-fold higher, suggesting increased sympathetic system activity. These results demonstrate that a KD can also increase BAT mitochondrial size and protein levels.

Microwave irradiation decreases ATP, increases free [Mg²âº], and alters in vivo intracellular reactions in rat brain.

Rapid inactivation of metabolism is essential for accurately determining the concentrations of metabolic intermediates in the in vivo state. We compared a broad spectrum of energetic intermediate metabolites and neurotransmitters in brains obtained by microwave irradiation to those obtained by freeze blowing, the most rapid method of extracting and freezing rat brain. The concentrations of many intermediates, cytosolic free NAD(P)(+) /NAD(P)H ratios, as well as neurotransmitters were not affected by the microwave procedure. However, the brain concentrations of ATP were about 30% lower, whereas those of ADP, AMP, and GDP were higher in the microwave-irradiated compared with the freeze-blown brains. In addition, the hydrolysis of approximately 1 µmol/g of ATP, a major in vivo Mg(2+) -binding site, was related to approximately five-fold increase in free [Mg(2+) ] (0.53 ± 0.07 mM in freeze blown vs. 2.91 mM ± 0.48 mM in microwaved brains), as determined from the ratio [citrate]/[isocitrate]. Consequently, many intracellular properties, such as the phosphorylation potential and the ∆G' of ATP hydrolysis were significantly altered in microwaved tissue. The determinations of some glycolytic and TCA cycle metabolites, the phosphorylation potential, and the ∆G' of ATP hydrolysis do not represent the in vivo state when using microwave-fixed brain tissue.