Regulation Of Metabolic Responses Research Articles

Plant GABA: Not Just a Metabolite.

γ-Aminobutyric acid (GABA) accumulates rapidly when plants are exposed to stress. Whether GABA accumulation represents the regulation of metabolism in response to stress or an adaptive response to mitigate stress is unknown. Genetic manipulation of GABA levels has revealed that GABA accumulation functions in defense against drought and insect herbivory.

Abstract 243: A Novel PRKAG2 Mutation (K475E): Infantile-onset Hypertrophic Cardiomyopathy Phenotype and Impact on mTOR Signaling

Objective: PRKAG2 encodes the 2 subunit isoform of the 5’ AMP-activated protein kinase (AMPK), a heterotrimeric enzyme with major roles in regulation of energy metabolism in response to cellular stress. Mutations in PRKAG2 have been implicated in a unique hypertrophic cardiomyopathy (HCM) characterized by cardiac glycogen overload and hypertrophy. We identified a novel PRKAG2 mutation in a neonate with prenatal onset of HCM noted on a 27-week prenatal ultrasound. Molecular testing for HCM revealed a de novo PRKAG2 mutation (K475E). We aim to investigate the signaling pathway of mutation in PRKAG2 gene. Methods: HEK-293 and H9C2 cells stably expressing wild type (WT) or K475E FLAG-tagged 2 from a tetracycline-inducible promoter were established. Basal AMPK activities were measured at varying AMP concentrations. Phenformin-stimulated AMPK activity and T172-phosphorylation were also measured. Changes in mTOR signaling pathway were accessed by Western blot. Cells were treated with angiotensin II, rapamycin (RAPA) or both followed by staining or flow cytometry to assess cell area/size. Primary fibroblasts from the K475E patient and non-diseased controls were cultured and compared. Finally, transgenic mice with cardiac-specific overexpression of human wild type (Tg WT ) or K475E (TgK 475E ) were established for further study of the PRKAG2 phenotype by histology and echocardiography. Result: HEK-293 cells: K475E mutation induced a markedly increase in the basal AMPK activity and T172-phosphorylation, reduced sensitivity to AMP in allosteric activation and loss of response to phenformin. H9c2 cells: K475E mutation induced increases in the phosphorylation of p-P70S6K, p-4EBP1 and a hypertrophy phenotype which was reversed by RAPA. Transgenic mice: Tg K475E mice had an early onset hypertrophy and were prone to sudden death. Histology revealed large vacuoles and high glycogen content in Tg K475E myocytes throughout the ventricles. Echocardiography confirmed significantly enlarged heart in Tg K475E mice. Conclusions: We identified a novel infantile-onset PRKAG2 mutation with a severe cardiac phenotype. In vitro study revealed involvement of mTOR signaling pathway and reversal of the hypertrophy phenotype by mTOR inhibitor.

Structural Evolution and Dynamics of the p53 Proteins.

The family of the p53 tumor suppressive transcription factors includes p73 and p63 in addition to p53 itself. Given the high degree of amino-acid-sequence homology and structural organization shared by the p53 family members, they display some common features (i.e., induction of cell death, cell-cycle arrest, senescence, and metabolic regulation in response to cellular stress) as well as several distinct properties. Here, we describe the structural evolution of the family members with recent advances on the molecular dynamic studies of p53 itself. A crucial role of the carboxy-terminal domain in regulating the properties of the DNA-binding domain (DBD) supports an induced-fit mechanism, in which the binding of p53 on individual promoters is preferentially regulated by the KOFF over KON.

Regulation of Galactolipid Biosynthesis by Overexpression of the Rice MGD Gene Contributes to Enhanced Aluminum Tolerance in Tobacco.

Membrane lipid alterations affect Al tolerance in plants, but little is known about the regulation of membrane lipid metabolism in response to Al stress. Transgenic tobacco (Nicotiana tabacum) overexpressing rice monogalactosyldiacylglycerol (MGDG) synthase (OsMGD) gene and wild-type tobacco plants were exposed to AlCl3, and the impact of Al toxicity on root growth, Al accumulation, plasma membrane integrity, lipid peroxidation and membrane lipid composition were investigated. Compared with the wild type, the transgenic plants exhibited rapid regrowth of roots after removal of Al and less damage to membrane integrity and lipid peroxidation under Al stress, meanwhile, the Al accumulation showed no difference between wild-type and transgenic plants. Lipid analysis showed that Al treatment dramatically decreased the content of MGDG and the ratio of MGDG to digalactosyldiacylglycerol (DGDG) in wild-type plants, while it was unchanged in transgenic plants. The stable of MGDG level and the ratio of MGDG/DGDG contribute to maintain the membrane stability and permeability. Moreover, Al caused a significant increase in phospholipids in wild-type plants, resulting in a high proportion of phospholipids and low proportion of galactolipids, but these proportions were unaffected in transgenic plants. The high proportion of phospholipids could contribute to a higher rate of Al3+ binding in the membrane and thereby leads to more membrane perturbation and damage. These results show that the regulation of galactolipid biosynthesis could play an important role in maintaining membrane structure and function under Al stress.

Environmental Statistics and Optimal Regulation

Any organism is embedded in an environment that changes over time. The timescale for and statistics of environmental change, the precision with which the organism can detect its environment, and the costs and benefits of particular protein expression levels all will affect the suitability of different strategies - such as constitutive expression or graded response - for regulating protein levels in response to environmental inputs. We propose a general framework - here specifically applied to the enzymatic regulation of metabolism in response to changing concentrations of a basic nutrient - to predict the optimal regulatory strategy given the statistics of fluctuations in the environment and measurement apparatus, respectively, and the costs associated with enzyme production. We use this framework to address three fundamental questions: (i) when a cell should prefer thresholding to a graded response; (ii) when there is a fitness advantage to implementing a Bayesian decision rule; and (iii) when retaining memory of the past provides a selective advantage. We specifically find that: (i) relative convexity of enzyme expression cost and benefit influences the fitness of thresholding or graded responses; (ii) intermediate levels of measurement uncertainty call for a sophisticated Bayesian decision rule; and (iii) in dynamic contexts, intermediate levels of uncertainty call for retaining memory of the past. Statistical properties of the environment, such as variability and correlation times, set optimal biochemical parameters, such as thresholds and decay rates in signaling pathways. Our framework provides a theoretical basis for interpreting molecular signal processing algorithms and a classification scheme that organizes known regulatory strategies and may help conceptualize heretofore unknown ones.

Environmental statistics and optimal regulation.

Any organism is embedded in an environment that changes over time. The timescale for and statistics of environmental change, the precision with which the organism can detect its environment, and the costs and benefits of particular protein expression levels all will affect the suitability of different strategies–such as constitutive expression or graded response–for regulating protein levels in response to environmental inputs. We propose a general framework–here specifically applied to the enzymatic regulation of metabolism in response to changing concentrations of a basic nutrient–to predict the optimal regulatory strategy given the statistics of fluctuations in the environment and measurement apparatus, respectively, and the costs associated with enzyme production. We use this framework to address three fundamental questions: (i) when a cell should prefer thresholding to a graded response; (ii) when there is a fitness advantage to implementing a Bayesian decision rule; and (iii) when retaining memory of the past provides a selective advantage. We specifically find that: (i) relative convexity of enzyme expression cost and benefit influences the fitness of thresholding or graded responses; (ii) intermediate levels of measurement uncertainty call for a sophisticated Bayesian decision rule; and (iii) in dynamic contexts, intermediate levels of uncertainty call for retaining memory of the past. Statistical properties of the environment, such as variability and correlation times, set optimal biochemical parameters, such as thresholds and decay rates in signaling pathways. Our framework provides a theoretical basis for interpreting molecular signal processing algorithms and a classification scheme that organizes known regulatory strategies and may help conceptualize heretofore unknown ones.

Implications from a pharmacogenomic analysis: Nerium oleander leaf distillate supplemented diet regulates cholesterol metabolism in rats

Context: Despite the usage of Nerium oleander L. (Apocynaceae) for anticancer studies and traditional remediation, the regulatory effect of N. oleander leaf distillate on cholesterol metabolism is not disclosed sufficiently.Objective: Cholesterol is an important biological molecule and the synthesis rate is regulated by the amount of cholesterol uptake from the diet. The aim of this study was to investigate the regulation of cholesterol metabolism in response to a high-fat diet (HFD) and the effects of N. oleander leaf distillate-supplemented diet (NOHFD) in rats.Materials and methods: Microarray technology was used to clarify the regulation of cholesterol mechanism in HFD and NOHFD-fed rats (375 μg/0.5 mL distilled water applied by gavage). The treatment period was 90 days. Rat liver tissues were used for microarray analysis using the Affymetrix GeneChip Rat Genome platform. Results of groups were statistically analyzed with the Partek 6.6 bioinformatic program.Results: The HFD group exhibited alterations in the expression levels of about 1945 genes with respect to the normal diet (ND) group. The results showed that expression levels of 47 genes were altered related to cholesterol metabolism in HFD and NOHFD groups. The expression levels of seven genes in the NOHFD group were significantly closer to those in the ND group than those of the HFD group.Discussion and conclusion: To conclude, findings suggest that N. oleander leaf distillate-supplemented food has considerable beneficial effects on cholesterol metabolism-related gene expression levels.

Fine-tuning Metabolic Switches

Fine-tuning Metabolic Switches

Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks.

Plants regularly face adverse growth conditions, such as drought, salinity, chilling, freezing, and high temperatures. These stresses can delay growth and development, reduce productivity, and, in extreme cases, cause plant death. Plant stress responses are dynamic and involve complex cross-talk between different regulatory levels, including adjustment of metabolism and gene expression for physiological and morphological adaptation. In this review, information about metabolic regulation in response to drought, extreme temperature, and salinity stress is summarized and the signalling events involved in mediating stress-induced metabolic changes are presented.

Metabolic regulation in Escherichia coli in response to culture environments via global regulators

One of the ultimate goal of systems biology is to realize a virtual cell system in the computer. If this could be attained, it might be possible, for example, to quantitatively predict the effects of a culture environment and/or the removal/inactivation of specific genes on the metabolism without conducting many experiments. Thus, it may be possible to design cells, e.g., for more efficient production of a specific metabolite. To achieve this, it is important to properly understand the metabolic regulation mechanism and to develop a robust model by incorporating gene-level regulation into the enzymatic reaction model with the integration of different levels of information. However, the metabolic regulation in response to the change in culture environment is itself not well understood. Here, we overview how the culture environment affects cell metabolism via global regulators with sigma factors, considering the effects of carbon, nitrogen, and phosphate sources as well as oxygen, temperature, pH, and nutrient stress, etc., on transcriptional regulation. A variety of controlled strategies for the specific stimuli imposed on the cell appear to exist, and some of the regulations are interconnected by gene level regulation. Quantitative modeling for these regulation mechanisms is critical for efficient metabolic engineering of a cell.

Physiology: alone at the bottom, alone at the top

Physiology: alone at the bottom, alone at the top

A histidine-kinase cheA gene of Pseudomonas pseudoalcaligens KF707 not only has a key role in chemotaxis but also affects biofilm formation and cell metabolism

A histidine-kinase cheA gene in Pseudomonas pseudoalcaligenes KF707 plays a central role in the regulation of metabolic responses as well as in chemotaxis. Non-chemotactic mutants harboring insertions into the cheA gene were screened for their ability to form biofilms in the Calgary biofilm device. Notably, ≥95% decrease in the number of cells attached to the polystyrene surface was observed in cheA mutants compared to the KF707 wild-type biofilm phenotype. The ability to form mature biofilms was restored to wild-type levels, providing functional copies of the KF707 cheA gene to the mutants. In addition, phenotype micro-arrays and proteomic analyses revealed that several basic metabolic activities and a few periplasmic binding proteins of cheA mutant cells differed compared to those of wild-type cells. These results are interpreted as evidence of a strong integration between chemotactic and metabolic pathways in the process of biofilm development by P. pseudoalcaligenes KF707.

LEPROT and LEPROTL1 cooperatively decrease hepatic growth hormone action in mice

Growth hormone (GH) is a major metabolic regulator that functions by stimulating lipolysis, preventing protein catabolism, and decreasing insulin-dependent glucose disposal. Modulation of hepatic sensitivity to GH and the downstream effects on the GH/IGF1 axis are important events in the regulation of metabolism in response to variations in food availability. For example, during periods of reduced nutrient availability, the liver becomes resistant to GH actions. However, the mechanisms controlling hepatic GH resistance are currently unknown. Here, we investigated the role of 2 tetraspanning membrane proteins, leptin receptor overlapping transcript (LEPROT; also known as OB-RGRP) and LEPROT-like 1 (LEPROTL1), in controlling GH sensitivity. Transgenic mice expressing either human LEPROT or human LEPROTL1 displayed growth retardation, reduced plasma IGF1 levels, and impaired hepatic sensitivity to GH, as measured by STAT5 phosphorylation and Socs2 mRNA expression. These phenotypes were accentuated in transgenic mice expressing both proteins. Moreover, gene silencing of either endogenous Leprot or Leprotl1 in H4IIE hepatocytes increased GH signaling and enhanced cell-surface GH receptor. Importantly, we found that both LEPROT and LEPROTL1 expression were regulated in the mouse liver by physiologic and pathologic changes in glucose homeostasis. Together, these data provide evidence that LEPROT and LEPROTL1 influence liver GH signaling and that regulation of the genes encoding these proteins may constitute a molecular link between nutritional signals and GH actions on body growth and metabolism.

An integrated view on the metabolic regulation in response to culture environments and the specific gene knockout in Escherichia coli

An integrated view on the metabolic regulation in response to culture environments and the specific gene knockout in Escherichia coli

Pattern of distribution of IGF-1 and EGF in pancreatic islets of type 2 diabetic patients

Growth factors such as insulin growth factor-1 (IGF-1) and epidermal growth factor (EGF) play important roles in the regulation of cell metabolism in response to different internal as well as external stimuli. It is not clear how Type 2 diabetes mellitus influences the expression of these growth factors in the endocrine pancreas. The localization of IGF-1 and EGF was analyzed to determine the impact of Type 2 diabetes on the pattern of distribution of these growth factors in pancreatic islet cells. Methods: Pancreatic tissue samples retrieved, during pancreatectomy for pancreatic tumour, from patients with and without Type 2 diabetes were paraffin embedded. The tissue samples were chosen from non-neoplastic, clear excisional, margins. The expression of IGF-1 and EGF was investigated using immunofluorescence techniques. Results: IGF-1- and EGF-immunoreactive cells were observed in many cells located in the central region of pancreatic islets. The percentage of islet cells expressing IGF-1 and EGF decreased significantly (P < 0.05) in diabetic patients compared to control. Conclusion: The number of human pancreatic islet cells expressing IGF-1 and EGF decreased significantly after the onset of Type 2 diabetes. These growth factors may play a role in the function of the endocrine pancreas and in the pathogenesis of the diabetes mellitus.

A proteomic approach to identify differentially expressed plasma proteins between the fed and prolonged fasted states

Prolonged fasting is characterized by consecutive phases, a short period of adaptation (phase 1), phase 2 (P2) characterized by fat oxidation, and phase 3 (P3) during which energy requirements are mostly derived from increased protein utilization. At this latter stage, food seeking behavior is induced. Very few circulating biomolecules have been identified that are involved in the response to prolonged fasting. To this end, rat plasma samples were compared by a proteomic approach, using 2-DE. The results revealed a selective variation of the levels of apolipoprotein A-IV, A-I, and E, haptoglobin, transthyretin, plasma retinol binding-protein, and vitamin D binding-protein in P2 and P3. The variations in protein levels were confirmed by ELISA. Changes in mRNA levels encoding these proteins did not systematically correlate well with protein concentrations, and tissue-specific regulation of mRNA expression was observed, underlining the complex metabolic regulation in response to food deprivation. In late fasting, the marked reduction of apolipoprotein A-IV levels could contribute to the alarm signal that triggers refeeding. The variations of the other differentially expressed proteins are more likely related to lipid metabolism and insulin signaling alterations.

Regulation of Metabolic Responses by Adipocyte/ Macrophage Fatty Acid–Binding Proteins in Leptin-Deficient Mice

Fatty acid-binding proteins (FABPs) are cytosolic fatty acid chaperones that play a critical role in systemic regulation of lipid and glucose metabolism. In animals lacking the adipocyte/macrophage FABP isoforms aP2 and mal1, there is strong protection against diet-induced obesity, insulin resistance, type 2 diabetes, fatty liver disease, and hypercholesterolemic atherosclerosis. On high-fat diet, FABP-deficient mice also exhibit enhanced muscle AMP-activated kinase (AMPK) and reduced liver stearoyl-CoA desaturase-1 (SCD-1) activities. Here, we performed a cross between aP2(-/-), mal1(-/-), and leptin-deficient (ob/ob) mice to elucidate the role of leptin action on the metabolic phenotype of aP2-mal1 deficiency. The extent of obesity in the ob/ob-aP2-mal1(-/-) mice was comparable with ob/ob mice. However, despite severe obesity, ob/ob-aP2-mal1(-/-) mice remained euglycemic and demonstrated improved peripheral insulin sensitivity. There was also a striking protection from liver fatty infiltration in the ob/ob-aP2-mal1(-/-) mice with strong suppression of SCD-1 activity. On the other hand, the enhanced muscle AMPK activity in aP2-mal1(-/-) mice was lost in the ob/ob background. These results indicated that both decreased body weight and enhanced muscle AMPK activity in aP2-mal1(-/-) mice are potentially leptin dependent but improved systemic insulin sensitivity and protection from liver fatty infiltration are largely unrelated to leptin action and that insulin-sensitizing effects of FABP deficiency are, at least in part, independent of its effects on total-body adiposity.

Metabolic Responses Induced by Serial Harvesting of Alfalfa Pasture Established on Amended Acid Soil

Alfalfa pasture has not been sustainable on the coastal plain of the United States because of its intolerance to soil acidity. This study examined the responses of alfalfa metabolism to differential amendments of acid soil and to serial harvesting. The soil was spatially amended with different quantities of flue gas desulfurization sludge and gypsum after liming to pH 7. The serial harvests oscillated the RNA synthetic activity of glutamate dehydrogenase (GDH) from an oxidized to a reduced state irrespective of the soil amendments. The amplitudes of the redox cycles changed from one harvest to the next, thus demonstrating improved regrowth persistence. The chlorophyll, hexose, nucleotide, and protein contents, and the fructose‐1, 6‐bisphosphatase activity, decreased and fluctuated inconsistently in the successive cuttings. Consideration of the metabolic responses per harvest showed that the alfalfa had optimized saccharide metabolism in the first harvest, optimized RNA metabolism in the second harvest, optimized saccharide and RNA metabolism in the third harvest, and depressed saccharide metabolism in the fourth harvest, thereby optimizing the regrowth potential of the alfalfa pasture. Sustainability of the pasture was conferred by the coordinate compensatory regulation of metabolism in response to the synergistic interaction between the differential amendments and the nitrogen (N) nutrient excreted by the alfalfa into the soil.

Yeast lipidomics: Transcriptional and metabolic regulation in response to inositol

Addition of inositol to the growth medium induces a rapid and profound alteration in the pattern of synthesis and turnover of the major membrane phospholipids in the yeast, Saccharomyces cerevisiae. Simultaneous genome-wide changes occur in the expression of several distinct sets of genes which respond to signals emanating from the endoplasmic reticulum (ER) (see Abstract 799, S. Jesch and S. Henry), the major site of phospholipid synthesis. To gain insight into potential signals responsible for these transcriptional patterns, we conducted a kinetic analysis of the changes in lipid composition and metabolism and gene expression following the addition of inositol. We now report that the introduction of inositol results in a rapid increase in the total cellular content of phosphatidylinositol (PI) accompanied by a rapid decrease in phosphatidylcholine (PC) content. Moreover, changes in the acyl chain length and degree of unsaturation among the various PC species were also observed. These alterations in PI and PC metabolism, in turn, generated altered patterns of utilization of their immediate precursors. The relationship of altered lipid metabolism and the correlated changes in transcription will be discussed in terms of potential signaling mechanisms. Overall, these results suggest that alterations in membrane lipid metabolism produce acute effects on global gene expression patterns and that the interplay among several distinct endoplasmic reticulum membrane-localized signaling pathways contributes to this response. This work is supported by NIH Grant GM-19629.

Function of Mammalian LKB1 and Ca2+/Calmodulin-dependent Protein Kinase Kinase α as Snf1-activating Kinases in Yeast

The Snf1/AMP-activated protein kinase (AMPK) family is important for metabolic regulation in response to stress. In the yeast Saccharomyces cerevisiae, the Snf1 kinase cascade comprises three Snf1-activating kinases, Pak1, Tos3, and Elm1. The only established mammalian AMPK kinase is LKB1. We show that LKB1 functions heterologously in yeast. In pak1Delta tos3Delta elm1Delta cells, LKB1 activated Snf1 catalytic activity and conferred a Snf(+) growth phenotype. Coexpression of STRADalpha and MO25alpha, which form a complex with LKB1, enhanced LKB1 function. Thus, the Snf1/AMPK kinase cascade is functionally conserved between yeast and mammals. Ca(2+)/calmodulin-dependent kinase kinase (CaMKK) shows more sequence similarity to Pak1, Tos3, and Elm1 than does LKB1. When expressed in pak1Delta tos3Delta elm1Delta cells, CaMKKalpha activated Snf1 catalytic activity, restored the Snf(+) phenotype, and also phosphorylated the activation loop threonine of Snf1 in vitro. These findings indicate that CaMKKalpha is a functional member of the Snf1/AMPK kinase family and support CaMKKalpha as a likely candidate for an AMPK kinase in mammalian cells. Analysis of the function of these heterologous kinases in yeast provided insight into the regulation of Snf1. When activated by LKB1 or CaMKKalpha, Snf1 activity was significantly inhibited by glucose, suggesting that a mechanism independent of the activating kinases can mediate glucose signaling in yeast. Finally, this analysis provided evidence that Pak1 functions in another capacity, besides activating Snf1, to regulate the nuclear enrichment of Snf1 protein kinase in response to carbon stress.