Changes In Metabolic Regulation Research Articles

Transcriptomic profiling of long non-coding RNAs and messenger RNAs in the liver of mice during Toxoplasma gondii infection

BackgroundToxoplasma gondii is an intracellular protozoan parasite that can infect a wide range of warm-blooded animals, including humans. It poses significant health risks, particularly in immunocompromised individuals and during pregnancy, leading to severe disease manifestations. The liver, being a crucial organ involved in immune response and metabolic regulation, plays a critical role in the host's defense against T. gondii infection.MethodsIn this study, we utilized RNA sequencing to investigate the expression profiles of long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) in the liver of mice infected with T. gondii. By employing this method, we obtained a comprehensive overview of the alterations in gene expression occurring in the liver during infection.ResultsBy comparing the infected groups to the control groups, we identified numerous differentially expressed lncRNAs DElncRNAs and DEmRNAs at two stages of infection. Specifically, at the acute infection stage, we found 628 DElncRNAs, and 6346 DEmRNAs. At the chronic infection stage, we identified 385 DElncRNAs and 2513 DEmRNAs. Furthermore, we identified 1959 commonly expressed DEmRNAs, including IL27, Nos2, and Cxcr2, across two infection stages. Enrichment and co-location analyses revealed pathways linked to immune and inflammatory responses during T. gondii infection. Notably, through co-location analysis, our analysis revealed several DElncRNAs, including Gm29156, Gm29157, and Gm28644, which are potentially implicated in the progression of liver inflammation induced by T. gondii. Additionally, functional enrichment analysis disclosed stage-specific characteristics of liver inflammation and immune response, alongside changes in metabolic regulation and immunosuppression pathways.ConclusionsOur findings provide valuable insights into the expression patterns of lncRNAs and mRNAs in the liver at different stages of T. gondii infection. We identified potential regulatory factors and pathways implicated in liver inflammation, thereby enhancing our understanding of the molecular mechanisms underlying liver inflammation and immune responses during T. gondii infection. These findings could contribute to the development of targeted therapeutic strategies for liver inflammation in the context of T. gondii infection.Graphical

Downregulation of Mitochondrial Complex II (MC II) in Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are a group of diseases of hematopoietic stem cells (HSCs) that occur in the aging population and following therapy for unrelated cancers. The standard of care for MDS involves hypomethylating agents (HMAs) that invariably result in resistance and disease progression. We previously determined the role of mitochondrial metabolism in MDS pathophysiology, whereby suppression of mitochondrial activity leads to the accumulation of oncometabolites that competitively inhibit α-ketoglutarate-dependent dioxygenases (2OGDDs). Consequently, hypermethylation of epigenomes and hypoxia-inducible factor 1A (HIF1A) activation ensues a pseudohypoxia state that drives MDS pathogenesis. Our recent informatics analyses of primary MDS samples identified a patient subset with high TGF-β signaling in which mitochondrial complex II (MC II) genes were downregulated. MC II, or succinate dehydrogenase (SDH) is the smallest complex of the oxidative phosphorylation system that oxidizes succinate to fumarate and thereby donates electrons to reduce FAD to FADH 2 and the ubiquinone pool. Its downregulation results in the accumulation of Krebs cycle intermediate metabolites that can act as antagonists for 2OGDDs. To understand whether SDH dysregulation alone can contribute to MDS pathobiology, we generated a doxycycline-inducible and reversible mouse model (SDH-KD) that expresses shRNAs against the SDH subunits (SDHA, B, and D) in HSCs. These mice showed a gradual buildup of intracellular succinate in c-Kit + bone marrow (BM) cells and the peripheral blood, and a corresponding decrease of fumarate levels after induction for 2 weeks, 4 weeks, and 2 months. We found that knocking down Sdh was sufficient to induce pancytopenia and trilineage dysplasia in SDH-KD mice, while intracellular iron granules that surround the nucleus of erythroblasts, similar to ring sideroblasts commonly observed in MDS patients were also detected in BM and spleen smears. Of note, removing doxycycline and restoring Sdh expression rescued macrocytic anemia among other MDS phenotypes. Furthermore, treating SDH-KD mice with HMAs restored mitochondrial respiration and reversed HIF1A activation through the upregulation of SDH subunits. Using another MDS model, MllPTD/WT/ Vav1-Cre/Runx1 Flox/Flox, we found that HMA-treated HSCs differentiate towards CD11b +Gr1 + mature myeloid cells and that this requires Sdh expression. In conclusion, our results indicate that pseudohypoxia- and TGF-β signaling-associated changes in metabolic regulation caused by SDH downregulation underlie MDS pathogenesis. Elucidating the role of MC II in MDS pathobiology may provide alternative targets that can be combined with HMA or luspatercept therapy for MDS.

SAT354 Molecular and Histological Characterization of a Guinea Pig Model of Mild Hyperandrogenemia

Abstract Disclosure: R.N. Tanori: None. J.E. Barboza Sagrero: None. K. Noll-Bader: None. S. Chandu: None. P.E. Chappell: None. C.V. Bishop: Consulting Fee; Self; Oviva, Oxford Pharmaceuticals Inc. There are several rodent models for polycystic ovarian syndrome (PCOS), but unlike primates, most rodents will not form a long-lived corpus luteum (CL) unless they experience cervico-vaginal stimulation/mating. Our research group has begun to determine if guinea pigs (Cavia porcellus), rodents with long-lived CL, are a suitable model for mild hyperandrogenemia, a risk factor for PCOS. The objective of this analysis was to determine the relative abundance of each follicle type within a cross-section of 20-week old guinea pig ovarian tissue from control and T-treated females after 3 months of androgen exposure. Ovarian tissues were collected from six guinea pigs treated with cholesterol implants (vehicle, n=4) or testosterone implants (n=3), which elevated androgen levels ∼2-fold above control females. Tissues were embedded in paraffin wax and cut into serial sections (5 μm) and placed onto glass slides (1 section/slide) for hematoxylin and eosin staining to identify follicle morphology within the ovarian tissue. Every fifth section was stained and analyzed using an ECHO Revolve microscope and follicles counted using ImageJ. Follicles of interest were primordial, primary, multi-layer, antral, and corpus luteum, and counts were normalized to ovarian area. All follicle types were found in all the ovaries imaged, as well as some corpora lutea. Multilayer follicles were significantly reduced by chronic exposure to mild hyperandrogenemia (p=0.01). These should provide needed baseline analyses for future experiments employing guinea pigs as a model of PCOS. In addition to histological analysis, we have begun quantifying gene expression changes for hormones and receptors throughout the reproductive axis. Preliminary studies using qPCR illustrate significantly elevated expression levels of hypothalamic kiss1, pituitary gnrhr, and ovarian fshr in T-treated females. No discernable differences were observed between treatment groups for kiss1r, gnrh, or fshb. Ongoing studies will explore differences in estrogen and androgen receptor expression throughout the reproductive axis, as well as any changes in metabolic regulators in liver and skeletal muscle that are associated with mild hyperandrogenemia in these animals. Presentation Date: Saturday, June 17, 2023

Hyperphosphatemia increases inflammation to exacerbate anemia and skeletal muscle wasting independently of FGF23-FGFR4 signaling.

Elevations in plasma phosphate concentrations (hyperphosphatemia) occur in chronic kidney disease (CKD), in certain genetic disorders, and following the intake of a phosphate-rich diet. Whether hyperphosphatemia and/or associated changes in metabolic regulators, including elevations of fibroblast growth factor 23 (FGF23) directly contribute to specific complications of CKD is uncertain. Here, we report that similar to patients with CKD, mice with adenine-induced CKD develop inflammation, anemia, and skeletal muscle wasting. These complications are also observed in mice fed high phosphate diet even without CKD. Ablation of pathologic FGF23-FGFR4 signaling did not protect mice on an increased phosphate diet or mice with adenine-induced CKD from these sequelae. However, low phosphate diet ameliorated anemia and skeletal muscle wasting in a genetic mouse model of CKD. Our mechanistic in vitro studies indicate that phosphate elevations induce inflammatory signaling and increase hepcidin expression in hepatocytes, a potential causative link between hyperphosphatemia, anemia, and skeletal muscle dysfunction. Our study suggests that high phosphate intake, as caused by the consumption of processed food, may have harmful effects irrespective of pre-existing kidney injury, supporting not only the clinical utility of treating hyperphosphatemia in CKD patients but also arguing for limiting phosphate intake in healthy individuals.

Changes in Metabolic Regulation and the Microbiota Composition after Supplementation with Different Fatty Acids in db/db Mice.

Introduction The effects of fatty acids on health vary and depend on the type, amount, and route of consumption. EPA and DHA have a defined role in health, unlike coconut oil. Objective The aim was to investigate the changes in metabolic regulation and the composition of the culture-dependent microbiota after supplementation with different fatty acids in db/db mice. Material and Methods. We were using 32 8-week-old db/db mice, supplemented for eight weeks with EPA/DHA derived from microalgae as well as coconut oil. The lipid, hormonal profiles, and composition of the culture-dependent microbiota and the phylogenetic analysis based on the 16S rRNA gene sequencing were determined for identification of the intestinal microbiota. Results Enriched diet with EPA/DHA reduced TNF-α, C-peptide, insulin resistance, resistin, and the plasma atherogenic index, but increased TC, LDL-c, VLDL-c, and TG without changes in HDL-c. Coconut oil raised the HDL-c, GIP, and TNF-α, with TG, insulin resistance, adiponectin, and C-peptide reduced. Conclusion The most abundant microbial populations were Firmicutes and the least Proteobacteria. EPA/DHA derived from microalgae contributes to improving the systemic inflammatory status, but depressed the diversity of the small intestine microbiota. Coconut oil only decreased the C-peptide, raising TNF-α, with an unfavorable hormonal and lipid profile.

Increased Reliance on Carbohydrates for Aerobic Exercise in Highland Andean Leaf-Eared Mice, but Not in Highland Lima Leaf-Eared Mice

Exercise is an important performance trait in mammals and variation in aerobic capacity and/or substrate allocation during submaximal exercise may be important for survival at high altitude. Comparisons between lowland and highland populations is a fruitful approach to understanding the mechanisms for altitude differences in exercise performance. However, it has only been applied in very few highland species. The leaf-eared mice (LEM, genus Phyllotis) of South America are a promising taxon to uncover the pervasiveness of hypoxia tolerance mechanisms. Here we use lowland and highland populations of Andean and Lima LEM (P. andium and P. limatus), acclimated to common laboratory conditions, to determine exercise-induced maximal oxygen consumption (O2max), and submaximal exercise metabolism. Lowland and highland populations of both species showed no difference in O2max running in either normoxia or hypoxia. When run at 75% of O2max, highland Andean LEM had a greater reliance on carbohydrate oxidation to power exercise. In contrast, highland Lima LEM showed no difference in exercise fuel use compared to their lowland counterparts. The higher carbohydrate oxidation seen in highland Andean LEM was not explained by maximal activities of glycolytic enzymes in the gastrocnemius muscle, which were equivalent to lowlanders. This result is consistent with data on highland deer mouse populations and suggests changes in metabolic regulation may explain altitude differences in exercise performance.

Metabolomics reveals the influences of smoke-water and karrikinolide on the biosynthesis of flavonoids and terpenoids in Salvia miltiorrhiza.

Metabolomics was used to study the influences of smoke-water (SW) and karrikinolide (KAR1) on the biosynthesis of flavonoids and terpenoids in Salvia miltiorrhiza Bunge. The results showed that a total of 178 and 199 differential metabolites were obtained in SW and KAR1, respectively, compared to the control. The differential metabolites were assigned to the corresponding metabolic pathways. The results indicated that some metabolic pathways in treatments of SW and KAR1 overlapped, suggesting that treatments of SW and KAR1 showed similar effects on the metabolic mechanism of S. miltiorrhiza. To obtain a clear overview of changes in metabolic regulation, TCA cycle, glycolytic pathway, biosynthesis of flavonoids and terpenoids and amino acids metabolism pathway were mapped into a network. We found that treatments with SW and KAR1 could significantly promote the biosynthesis of flavonoids and terpenoids in S. miltiorrhiza. This study could help us better understand the influences of SW and KAR1 on secondary metabolites and their underlying mechanism.

Early Intervention in Psychosis: Effectiveness and Implementation of a Combined Exercise and Health Behavior Intervention Within Routine Care.

AimYoung people with psychosis have higher rates of obesity, premature cardiovascular disease, and death compared to non-psychotic peers in the general population due to changes in metabolic regulation linked to antipsychotic medication and adverse health risk behaviors. The aim of this paper is to outline the development, implementation, and evaluation of a combined 12-week exercise and health behavior intervention delivered as part of an Early Intervention in Psychosis (EIP) routine service, within the UK.MethodsParticipants (n = 27) completed a 12-week combined intervention program, engaging in weekly, 90-min sessions comprising a healthy behavior education session (45 min), followed by a facilitated exercise session (45 min). Anthropometric data from participants (n = 26) were collected at baseline, 12 weeks, and 12 months post-intervention. Health behaviors and clinical measurements were assessed at baseline and 12 months.ResultsMean baseline data suggests participants were at an increased health risk on entry to the program, with elevated values in mean body mass index (BMI; 70% overweight/obese), waist circumference, resting heart rate, and triglycerides. Fifty percent reported smoking daily, 64% ate < 5 fruits/vegetables per day, and 52% of participants were prescribed highly obesogenic antipsychotic medications (i.e., Olanzapine). At 12 weeks and 12 months, no changes were observed in mean BMI, waist circumference or any other clinical variable (p > 0.05). At 12 months, participants reported a positive impact on health behaviors including improved diet, increased physical activity levels, and cessation of substance use (n = 2), alcohol use (n = 2), and smoking (n = 4). Focus groups captured participant experiences, engagement with and satisfaction with the program, including challenges/barriers to program adherence.ConclusionsThe 12-week exercise and health behaviors program supported participants to attenuate their physical health risk which was sustained at 12-month follow-up. Self-reported positive health behavior changes are likely to have contributed to the prevention of excessive weight gain in this high-risk period. The evaluation was designed to have validity for a “real world EIP setting” and reflect the complexity of delivery to this participant group. Evaluation findings influenced subsequent commissioning of the physical health intervention as an ongoing element of routine EIP care within the participant site.

High fat diet consumption results in mitochondrial dysfunction, oxidative stress, and oligodendrocyte loss in the central nervous system

Metabolic syndrome is a key risk factor and co-morbidity in multiple sclerosis (MS) and other neurological conditions, such that a better understanding of how a high fat diet contributes to oligodendrocyte loss and the capacity for myelin regeneration has the potential to highlight new treatment targets. Results demonstrate that modeling metabolic dysfunction in mice with chronic high fat diet (HFD) consumption promotes loss of oligodendrocyte progenitors across the brain and spinal cord. A number of transcriptomic and metabolomic changes in ER stress, mitochondrial dysfunction, and oxidative stress pathways in HFD-fed mouse spinal cords were also identified. Moreover, deficits in TCA cycle intermediates and mitochondrial respiration were observed in the chronic HFD spinal cord tissue. Oligodendrocytes are known to be particularly vulnerable to oxidative damage, and we observed increased markers of oxidative stress in both the brain and spinal cord of HFD-fed mice. We additionally identified that increased apoptotic cell death signaling is underway in oligodendrocytes from mice chronically fed a HFD. When cultured under high saturated fat conditions, oligodendrocytes decreased both mitochondrial function and differentiation. Overall, our findings show that HFD-related changes in metabolic regulators, decreased mitochondrial function, and oxidative stress contribute to a loss of myelinating cells. These studies identify HFD consumption as a key modifiable lifestyle factor for improved myelin integrity in the adult central nervous system and in addition new tractable metabolic targets for myelin protection and repair strategies.

Molecular Mechanism of Transcriptional Regulation by Chromatin Reader Transcription Factor 19 (TCF19) during Metabolic Stress

Changing nutritional environment confers epigenetic memories that often pre‐dispose the human body to certain metabolic disorders. Histone post‐translational modifications (PTMs), and their selective recognition by reader domain containing proteins constitute a transcription regulatory network that helps in maintaining cellular metabolic homeostasis, although the exact mechanism is still unclear. Here we have studied a novel glucose sensing and endoplasmic reticulum (ER) stress‐responsive transcription regulator, TCF19, which regulates transcription of key metabolic and stress‐responsive genes through recognition of histone PTMs. Through biochemical and biophysical techniques, we have found that TCF19 Plant Homeodomain (PHD) finger has a unique preference for the lysine 4 trimethylation of histone H3, which is a transcription activation modification. Using siRNA mediated TCF19 depletion in human hepatoma cell line HepG2 and primary human hepatocyte‐like cell HepaRG, we observed a global effect on metabolic pathways and interestingly the gluconeogenic genes were significantly upregulated. Through endogenous immunoprecipitation, we found that TCF19 interacts with components of a histone deacetylase complex NuRD (Nucleosome Remodelling and Deacetylase) and Chromatin immunoprecipitation studies indicate their co‐recruitment onto promoters of gluconeogenic genes under high glucose conditions, suggesting that the observed repression is possibly mediated in concert with NuRD complex. In‐depth analysis revealed that TCF19 also represses gluconeogenesis in response to insulin stimulation in both HepG2 and HepaRG cells. Deletion mutants of TCF19 lacking the PHD finger were unable to repress gluconeogenesis in response to insulin, underscoring the importance of histone modification mediated regulation of insulin action. We have also observed that TCF19 associates with several other transcription regulators of metabolic genes which have an important role in ER stress and cell survival and has been widely linked to type 2 diabetes progression. Overall, our study presents a novel transcription regulator that integrates metabolic (glucose), as well as hormonal (insulin) signal. These findings seek to expand our understanding of the role of epigenetic changes in metabolic regulation, and in the long run, provide an opportunity for therapeutic intervention to treat metabolic disorders like diabetes.Support or Funding InformationThe study was supported by research grant entitled Biomolecular Assembly, Recognition and Dynamics (BARD) (Grant 12‐R&amp;D‐SIN‐5.04‐0103) by the Department of Atomic Energy (DAE), Govt. of IndiaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Shift in Food Intake and Changes in Metabolic Regulation and Gene Expression during Simulated Night-Shift Work: A Rat Model.

Night-shift work is linked to a shift in food intake toward the normal sleeping period, and to metabolic disturbance. We applied a rat model of night-shift work to assess the immediate effects of such a shift in food intake on metabolism. Male Wistar rats were subjected to 8 h of forced activity during their rest (ZT2-10) or active (ZT14-22) phase. Food intake, body weight, and body temperature were monitored across four work days and eight recovery days. Food intake gradually shifted toward rest-work hours, stabilizing on work day three. A subgroup of animals was euthanized after the third work session for analysis of metabolic gene expression in the liver by real-time polymerase chain reaction (PCR). Results show that work in the rest phase shifted food intake to rest-work hours. Moreover, liver genes related to energy storage and insulin metabolism were upregulated, and genes related to energy breakdown were downregulated compared to non-working time-matched controls. Both working groups lost weight during the protocol and regained weight during recovery, but animals that worked in the rest phase did not fully recover, even after eight days of recovery. In conclusion, three to four days of work in the rest phase is sufficient to induce disruption of several metabolic parameters, which requires more than eight days for full recovery.

A comparison of chronic AICAR treatment-induced metabolic adaptations in red and white muscles of rats.

The signaling molecule 5'-AMP-activated protein kinase plays a pivotal role in metabolic adaptations. Treatment with 5-aminoimidazole-4-carboxamide-1-β-D-ribofranoside (AICAR) promotes the expression of metabolic regulators and components involved in glucose uptake, mitochondrial biogenesis, and fatty acid oxidation in skeletal muscle cells. Our aim was to determine whether AICAR-induced changes in metabolic regulators and components were more prominent in white or red muscle. Rats were treated with AICAR (1 mg/g body weight/day) for 14 days, resulting in increased expression levels of nicotinamide phosphoribosyltransferase (NAMPT), peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), glucose transporter 4 proteins, and enhanced mitochondrial biogenesis. These changes were more prominent in white rather than red gastrocnemius muscle or were only observed in the white gastrocnemius. Our results suggest that AICAR induces the expression of metabolic regulators and components, especially in type II (B) fibers.

Thermal manipulation during embryogenesis has long-term effects on muscle and liver metabolism in fast-growing chickens.

Fast-growing chickens have a limited ability to tolerate high temperatures. Thermal manipulation during embryogenesis (TM) has previously been shown to lower chicken body temperature (Tb) at hatching and to improve thermotolerance until market age, possibly resulting from changes in metabolic regulation. The aim of this study was to evaluate the long-term effects of TM (12 h/d, 39.5°C, 65% RH from d 7 to 16 of embryogenesis vs. 37.8°C, 56% RH continuously) and of a subsequent heat challenge (32°C for 5 h at 34 d) on the mRNA expression of metabolic genes and cell signaling in the Pectoralis major muscle and the liver. Gene expression was analyzed by RT-qPCR in 8 chickens per treatment, characterized by low Tb in the TM groups and high Tb in the control groups. Data were analyzed using the general linear model of SAS considering TM and heat challenge within TM as main effects. TM had significant long-term effects on thyroid hormone metabolism by decreasing the muscle mRNA expression of deiodinase DIO3. Under standard rearing conditions, the expression of several genes involved in the regulation of energy metabolism, such as transcription factor PGC-1α, was affected by TM in the muscle, whereas for other genes regulating mitochondrial function and muscle growth, TM seemed to mitigate the decrease induced by the heat challenge. TM increased DIO2 mRNA expression in the liver (only at 21°C) and reduced the citrate synthase activity involved in the Krebs cycle. The phosphorylation level of p38 Mitogen-activated-protein kinase regulating the cell stress response was higher in the muscle of TM groups compared to controls. In conclusion, markers of energy utilization and growth were either changed by TM in the Pectoralis major muscle and the liver by thermal manipulation during incubation as a possible long-term adaptation limiting energy metabolism, or mitigated during heat challenge.

Parameter optimization and sensitivity analysis for large kinetic models using a real-coded genetic algorithm

Dynamic modeling is a powerful tool for predicting changes in metabolic regulation. However, a large number of input parameters, including kinetic constants and initial metabolite concentrations, are required to construct a kinetic model. Therefore, it is important not only to optimize the kinetic parameters, but also to investigate the effects of their perturbations on the overall system. We investigated the efficiency of the use of a real-coded genetic algorithm (RCGA) for parameter optimization and sensitivity analysis in the case of a large kinetic model involving glycolysis and the pentose phosphate pathway in Escherichia coli K-12. Sensitivity analysis of the kinetic model using an RCGA demonstrated that the input parameter values had different effects on model outputs. The results showed highly influential parameters in the model and their allowable ranges for maintaining metabolite-level stability. Furthermore, it was revealed that changes in these influential parameters may complement one another. This study presents an efficient approach based on the use of an RCGA for optimizing and analyzing parameters in large kinetic models.

Plasma Leptin, Ghrelin and Indexes of Glucose and Lipid Metabolism in Relation to the Appearance of Post‐Weaning Oestrus in Mediterranean Obese Sows (Iberian Pig)

Iberian pig is the most abundant Mediterranean swine. The lack of knowledge of the reproductive physiology of Mediterranean genotypes, with predisposition to obesity, led us to evaluate the influence of body condition and metabolic status at weaning on the resumption of follicular growth and the appearance of post-weaning oestrus. Females failing to display post-weaning oestrus showed a high decrease in backfat mass during lactation; backfat depth at weaning was therefore lower than in sows becoming in oestrus. Females not bearing oestrus behaviour showed lower plasma leptin levels and higher ghrelin concentrations at weaning. Moreover, these sows evidenced dyslipidemic profile (increased triglyceridemia and cholesterolemia) and mobilization of fat reserves. Hence, changes in metabolic regulation of Iberian pigs may originate large effects on the resumption of ovulatory activity after weaning.

Morphine dependence and withdrawal: changes in metabolic regulation and behavior

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Proteomic and metabolic characterization of aCandida albicansmutant resistant to the antimicrobial peptide MUC7 12-mer

MUC7 12-mer is a cationic peptide derived from the N-terminal portion of human mucin MUC7, exhibiting potent antibacterial and antifungal properties. To advance our knowledge regarding the mechanisms of action of MUC7 peptide against an opportunistic fungal pathogen Candida albicans, we sought to develop and characterize mutant(s) resistant to this peptide. One of the selected mutants, designated #37, was much less susceptible to the MUC7 12-mer in a killing assay than the parental strain (ED(50)>40 vs. c. 6 microM, respectively). Difference gel electrophoresis (DIGE) analysis of the mutant revealed elevation of several glycolytic enzymes. The mutant also exhibited lowered ATP contents along with a relatively lower rate of oxygen consumption, as well as inability to grow on nonfermentable carbon sources. These properties are likely to be associated with changes in metabolic regulation, rather than lack of functional mitochondria, as determined by rhodamine 123 staining. Analysis of interaction between fluorescently labeled peptide and cells of both strains revealed that resistance of the mutant #37 is associated with changes in the process of transition between surface-bound state of the peptide to its internalization marking cell death.

Transcriptomic analysis of extensive changes in metabolic regulation in Kluyveromyces lactis strains.

Genome-wide analysis of transcriptional regulation is generally carried out on well-characterized reference laboratory strains; hence, the characteristics of industrial isolates are therefore overlooked. In a previous study on the major cheese yeast Kluyveromyces lactis, we have shown that the reference strain and an industrial strain used in cheese making display a differential gene expression when grown on a single carbon source. Here, we have used more controlled conditions, i.e., growth in a fermentor with pH and oxygen maintained constant, to study how these two isolates grown in glucose reacted to an addition of lactose. The observed differences between sugar consumption and the production of various metabolites, ethanol, acetate, and glycerol, correlated with the response were monitored by the analysis of the expression of 482 genes. Extensive differences in gene expression between the strains were revealed in sugar transport, glucose repression, ethanol metabolism, and amino acid import. These differences were partly due to repression by glucose and another, yet-unknown regulation mechanism. Our results bring to light a new type of K. lactis strain with respect to hexose transport gene content and repression by glucose. We found that a combination of point mutations and variation in gene regulation generates a biodiversity within the K. lactis species that was not anticipated. In contrast to S. cerevisiae, in which there is a massive increase in the number of sugar transporter and fermentation genes, in K. lactis, interstrain diversity in adaptation to a changing environment is based on small changes at the level of key genes and cell growth control.

Methyl jasmonate down‐regulates endogenous cytokinin levels in cotyledons of Cucurbita pepo (zucchini) seedlings

Endogenous cytokinins (CK) were measured in Cucurbita pepo L. (zucchini) cotyledons after spraying 7‐day‐old seedlings with methyl jasmonate (MeJA) to examine whether the senescence‐promoting action of MeJA is associated with changes in metabolic regulation of CK levels. MeJA promoted senescence as estimated by the loss of chlorophyll and injury to chloroplast ultrastructure. This was already detected one day after treatment. The contents of physiologically active CKs determined by high‐performance liquid chromatography‐mass spectrometry (CK bases: trans‐zeatin, dihydrozeatin and isopentenyladenine and their ribosides), especially trans‐zeatin and its riboside declined considerably. CK nucleotides and physiologically inactive CK 7‐ and 9‐glucosides were also markedly decreased whereas the content of storage CK O‐glucosides as well as cis‐isomers of zeatin declined only slightly. Chlorophyll content and the normal chloroplast structure recovered 5 days after the MeJa treatment. The process of photosynthetic apparatus recovery correlated with an increased concentration of physiologically active CKs, especially Z. In contrast, the content of CK nucleotides and storage CK O‐glucosides decreased further. We suggest that the ability of MeJA to promote some aspects of senescence in intact zucchini cotyledons is at least partially due to down‐regulation of endogenous CK levels and their interconversion between active and inactive forms.

Juvenile diet restriction and the aging and reproduction of adult Drosophila melanogaster.

Mutations of the insulin signal pathway in Drosophila melanogaster produce long-lived adults with many correlated phenotypes. Homozygotes of insulin-like receptor (InR) and insulin-like receptor substrate (chico) delay time to eclosion, reduce body size, decrease reproduction and increase life span. Because these mutations are expressed through all life stages it is unclear when insulin signals must be reduced to increase life span. As a first analysis of this problem in D. melanogaster we have manipulated the larval diet to determine if changes in metabolic regulation at this stage are sufficient to slow aging. We controlled the dietary yeast fed to third instar larvae and studied the size, mortality, fecundity and hormones of the resulting adults, which were fed a normal, yeast-replete diet. Adults from yeast-deprived larvae phenocopied many traits of InR and chico mutants: small body size, delayed eclosion, reduced ovariole number and reduced age-specific fecundity. But unlike constitutive mutants of the insulin/IGF system, adults from yeast-deprived larvae had normal patterns of demographic senescence, and this was accompanied by normal insulin-like peptide and juvenile hormone syntheses. Surprisingly, the normal aging in these adults was also associated with greatly reduced fecundity. Although nutritional conditions of the larvae can affect the subsequent body size and fecundity of adults, these are not sufficient to slow aging.