Genomic and biochemical investigation of soybean antioxidant metabolism in response to growth at elevated carbon dioxide and elevated ozone

Metabolism in an environment containing of 21% oxygen has a high risk of oxidative damage due to the formation of reactive oxygen species. Therefore, plants have evolved an antioxidant system consisting of metabolites and enzymes that either directly scavenge ROS or recycle the antioxidant metabolites. Ozone is a temporally dynamic molecule that is both naturally occurring as well as an environmental pollutant that is predicted to increase in concentration in the future as anthropogenic precursor emissions rise. It has been hypothesized that any elevation in ozone concentration will cause increased oxidative stress in plants and therefore enhanced subsequent antioxidant metabolism, but evidence for this response is variable. Along with increasing atmospheric ozone concentrations, atmospheric carbon dioxide concentration is also rising and is predicted to continue rising in the future. The effect of elevated carbon dioxide concentrations on antioxidant metabolism varies among different studies in the literature. Therefore, the question of how antioxidant metabolism will be affected in the most realistic future atmosphere, with increased carbon dioxide concentration and increased ozone concentration, has yet to be answered, and is the subject of my thesis research. First, in order to capture as much of the variability in the antioxidant system as possible, I developed a suite of high-throughput quantitative assays for a variety of antioxidant metabolites and enzymes. I optimized these assays for Glycine max (soybean), one of the most important food crops in the world. These assays provide accurate, rapid and high-throughput measures of both the general and specific antioxidant action of plant tissue extracts. Second, I investigated how growth at either elevated carbon dioxide concentration or chronic elevated ozone concentration altered antioxidant metabolism, and the ability of soybean to respond to an acute oxidative stress in a controlled environment study. I found that growth at chronic elevated ozone concentration increased the antioxidant capacity of leaves, but was unchanged or only slightly increased following an acute oxidative stress, suggesting that growth at chronic elevated ozone concentration primed the antioxidant system. Growth at high carbon dioxide concentration decreased the antioxidant capacity of leaves, increased the response of the existing antioxidant enzymes to an acute oxidative stress, but dampened and delayed the transcriptional response, suggesting an entirely different regulation of the antioxidant system. Third, I tested the findings from the controlled environment study in a field setting by investigating the response of the soybean antioxidant system to growth at elevated carbon dioxide concentration, chronic elevated ozone concentration and the combination of elevated carbon dioxide concentration and elevated ozone concentration. In this study, I confirmed that growth at elevated carbon dioxide concentration decreased specific components of antioxidant metabolism in the field. I also verified that increasing ozone concentration is highly correlated with increases in the metabolic and genomic components of antioxidant metabolism, regardless of carbon dioxide concentration environment, but that the response to increasing ozone concentration was dampened at elevated carbon dioxide concentration. In addition, I found evidence suggesting an up regulation of respiratory metabolism at higher ozone concentration, which would supply energy and carbon for detoxification and repair of cellular damage. These results consistently support the conclusion that growth at elevated carbon dioxide concentration decreases antioxidant metabolism while growth at elevated ozone concentration increases antioxidant metabolism.

The regulation of AMP-activated protein kinase by vascular endothelial growth factor

The function of the vascular endothelium is to maintain vascular homeostasis, by providing an anti-thrombotic, anti-inflammatory and vasodilatory interface between circulating blood and the vessel wall, meanwhile facilitating the selective passage of blood components such as signaling molecules and immune cells. Dysfunction of the vascular endothelium is implicated in a number of pathological states including atherosclerosis and hypertension, and is thought to precede atherogenesis by a number of years. Vascular endothelial growth factor A (VEGF) is a crucial mitogenic signaling molecule, not only essential for embryonic development, but also in the adult for regulating both physiological and pathological angiogenesis. Previous studies by our laboratory have demonstrated that VEGF-A activates AMP-activated protein kinase (AMPK), the downstream component of a signaling cascade important in the regulation of whole body and cellular energy status. Furthermore, studies in our laboratory have indicated that AMPK is essential for VEGF-A-stimulated vascular endothelial cell proliferation. AMPK activation typically stimulates anabolic processes and inhibits catabolic processes including cell proliferation, with the ultimate aim of redressing energy imbalance, and as such is an attractive therapeutic target for the treatment of obesity, metabolic syndromes, and type 2 diabetes. Metabolic diseases are associated with adverse cardiovascular outcomes and AMPK activation is reported to have beneficial effects on the vascular endothelium. The mechanism by which VEGF-A stimulates AMPK, and the functional consequences of VEGF-A-stimulated AMPK activation remain uncertain. The present study therefore aimed to identify the specific mechanism(s) by which VEGF-A regulates the activity of AMPK in endothelial cells, and how this might differ from the activation of AMPK by other agents. Furthermore, the role of AMPK in the pro-proliferative actions of VEGF-A was further examined. Human aortic and umbilical vein endothelial cells were therefore used as a model system to characterise the specific effect(s) of VEGF-A stimulation on AMPK activation. The present study reports that AMPK α1 containing AMPK complexes account for the vast majority of both basal and VEGF-A-stimulated AMPK activity. Furthermore, AMPK α1 is localized to the endoplasmic reticulum when sub-confluent, but translocated to the Golgi apparatus when cells are cultured to confluence. AMPK α2 appears to be associated with a structural cellular component, but neither α1 nor α2 complexes appear to translocate in response to VEGF-A stimulation. The present study confirms previous reports that when measured using the MTS cell proliferation assay, AMPK is required for VEGF-A-stimulated endothelial cell proliferation. However, parallel experiments measuring cell proliferation using the Real-Time Cell Analyzer xCELLigence system, do not agree with these previous reports, suggesting that AMPK may in fact be required for an aspect of mitochondrial metabolism which is enhanced by VEGF-A. Studies into the mitochondrial activity of endothelial cells have proved inconclusive at this time, but further studies into this are warranted. During previous studies in our laboratory, it was suggested that VEGF-A-stimulated AMPK activation may be mediated via the diacylglycerol (DAG)-sensitive transient receptor potential cation channel (TRPCs -3, -6 or -7) family of ion channels. The present study can neither confirm, nor exclude the expression of TRPCs in vascular endothelial cells, nor rule out their involvement in VEGF-A-stimulated AMPK activation; more specific investigative tools are required in order to characterise their involvement. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP)-stimulated Ca2+ release from acidic intracellular organelles is not required for AMPK activation by VEGF-A. Despite what is known about the mechanisms by which AMPK is activated, far less is known concerning the downregulation of AMPK activity, as observed in human and animal models of metabolic disease. Phosphorylation of AMPK α1 Ser485 (α2 Ser491) has recently been characterised as a mechanism by which the activity of AMPK is negatively regulated. We report here for the first time that VEGF-A stimulates AMPK α1 Ser485 phosphorylation independently of the previously reported AMPK α1 Ser485 kinases Akt (protein kinase B) and ERK1/2 (extracellular signal-regulated kinase 1/2). Furthermore, inhibition of protein kinase C (PKC), the activity of which is reported to be elevated in metabolic disease, attenuates VEGF-A- and phorbol 12-myristate 13-acetate (PMA)-stimulated AMPK α1 Ser485 phosphorylation, and increases basal AMPK activity. In contrast to this, PKC activation reduces AMPK activity in human vascular endothelial cells. Attempts to identify the PKC isoform responsible for inhibiting AMPK activity suggest that it is one (or more) of the Ca2+-regulated DAG-sensitive isoforms of PKC, however cross regulation of PKC isoform expression has limited the present study. Furthermore, AMPK α1 Ser485 phosphorylation was inversely correlated with human muscle insulin sensitivity. As such, enhanced AMPK α1 Ser485 phosphorylation, potentially mediated by increased PKC activation may help explain some of the reduced AMPK activity observed in metabolic disease.

Transcriptional regulation os phenylalaline biosynthesis and utilization

Conifer trees divert large quantities of carbon into the biosynthesis of phenylpropanoids, particularly to generate lignin, an important constituent of wood. Since phenylalanine is the precursor for phenylpropanoid biosynthesis, the precise regulation of phenylalanine synthesis and utilization should occur simultaneously. This crucial pathway is finely regulated primarily at the transcriptional level. Transcriptome analyses indicate that the transcription factors (TFs) preferentially expressed during wood formation in plants belong to the MYB and NAC families. Craven-Bartle et al. (2013) have shown in conifers that Myb8 is a candidate regulator of key genes in phenylalanine biosynthesis involved in the supply of the phenylpropane carbon skeleton necessary for lignin biosynthesis. This TF is able to bind AC elements present in the promoter regions of these genes to activate transcription. Constitutive overexpression of Myb8 in white spruce increased secondary-wall thickening and led to ectopic lignin deposition (Bomal et al. 2008). In Arabidopsis, the transcriptional network controlling secondary cell wall involves NAC-domain regulators operating upstream Myb transcription factors. Functional orthologues of members of this network described have been identified in poplar and eucalyptus, but in conifers functional evidence had only been obtained for MYBs. We have identified in the P. pinaster genome 37 genes encoding NAC proteins, which 3 NAC proteins could be potential candidates to be involved in vascular development (Pascual et al. 2015). The understanding of the transcriptional regulatory network associated to phenylpropanoids and lignin biosynthesis in conifers is crucial for future applications in tree improvement and sustainable forest management. This work is supported by the projects BIO2012-33797, BIO2015-69285-R and BIO-474 References: Bomal C, et al. (2008) Involvement of Pinus taeda MYB1 and MYB8 in phenylpropanoid metabolism and secondary cell wall biogenesis: a comparative in planta analysis. J Exp Bot. 59: 3925-3939. Craven-Bartle B, et al. (2013) A Myb transcription factor regulates genes of the phenylalanine pathway in maritime pine. Plant J, 74: 755-766. Pascual MB, et al. (2015) The NAC transcription factor family in maritime pine (Pinus pinaster): molecular regulation of two genes involved in stress responses. BMC Plant Biol, 15: 254.

Studying the role of the strawberry Fra protein family in the flavonoid metabolism during fruit ripening

Strawberry fruits are highly appreciated worldwide due to their pleasant flavor and aroma and to the health benefits associated to their consumption. An important part of these properties is due to their content in secondary metabolites, especially phenolic compounds, of which flavonoids are the most abundant in the strawberry fruit. Although the flavonoid biosynthesis pathway is uncovered, little is known about its regulation. The strawberry Fra a (Fra) genes constitute a large family of homologs of the major birch pollen allergen Bet v 1 and for which no equivalents exist in Arabidopsis. Our group has shown that Fra proteins are involved in the formation of colored compounds in strawberries (Muñoz et al., 2010), which mainly depends on the production of certain flavonoids; that they are structurally homologs to the PYR/PYL/RCAR Arabidopsis ABA receptor, and that they are able to bind flavonoids (Casañal et al., 2013). With these previous results, our working hypothesis is that the Fra proteins are involved in the regulation of the flavonoids pathway. They would mechanistically act as the ABA receptor, binding a protein interactor and a ligand to regulate a signaling cascade and/or act as molecular carriers. The main objective of this research is to characterize the Fra family in strawberry and gain insight into their role in the flavonoid metabolism. By RNAseq expression analysis in ripening fruits we have identified transcripts for 10 members of the Fra family. Although expressed in all tissues analyzed, each family member presents a unique pattern of expression, which suggests functional specialization for each Fra protein. Then, our next approach was to identify the proteins that interact with Fras and their ligands to gain knowledge on the role that these proteins play in the flavonoids pathway. To identify the interacting partners of Fras we have performed a yeast two hybrid (Y2H) screening against cDNA libraries of strawberry fruits at the green and red stages. A protein that shares a 95% homology to the Heat stress transcription factor A-4-C like of Fragaria vesca (HSA4C) interacts specifically with Fra1 and not with other family members, which suggests functional diversification of Fra proteins in specific signaling pathways. The Y2H screening is not yet saturated, so characterization of other interacting proteins with other members of the Fra family will shed light on the functional diversity within this gene family. This research will contribute to gain knowledge on how the flavonoid pathway, and hence, the fruit ripening, is regulated in strawberry; an economically important crop but for which basic research is still very limited. References: Muñoz, C, et al. (2010). The Strawberry Fruit Fra a Allergen Functions in Flavonoid Biosynthesis. Molecular Plant, 3(1): 113–124. Casañal, A, et al (2013). The Strawberry Pathogenesis-related 10 (PR-10) Fra a Proteins Control Flavonoid Biosynthesis by Binding Metabolic Intermediates. Journal of Biological Chemistry, 288(49): 35322–35332.

Starch metabolism in Leucoagaricus gongylophorus, the symbiotic fungus of leaf-cutting ants

Leucoagaricus gongylophorus, the symbiotic fungus of the leaf-cutting ants, degrades starch, this degradation being supposed to occur in the plant material which leafcutters forage to the nests, generating most of the glucose which the ants utilize for food. In the present investigation, we show that laboratory cultures of L. gongylophorus produce extracellular alpha-amylase and maltase which degrade starch to glucose, reinforcing that the ants can obtain glucose from starch through the symbiotic fungus. Glucose was found to repress a-amylase and, more severely, maltase activity, thus repressing starch degradation by L. gongylophorus, so that we hypothesize that: (1) glucose down-regulation of starch degradation also occurs in the Atta sexdens fungus garden; (2) glucose consumption from the fungus garden by A. sexdens stimutates degradation of starch from plant material by L. gongylophorus, which may represent a mechanism by which Leafcutters can control enzyme production by the symbiotic fungus. Since glucose is found in the fungus garden inside the nests, down-regulation of starch degradation by glucose is supposed to occur in the nest and play a part in the control of fungal enzyme production by leafcutters. (c) 2005 Elsevier GmbH. All rights reserved.

Long term betaine supplementation regulates genes involved in lipid and cholesterol metabolism of two muscles from an obese pig breed

This study evaluates the effects of betaine supplementation (1 g kg−1 for 20 weeks) on the regulation of genes involved in lipid and cholesterol metabolism of Longissimus lumborum and Biceps femoris from obese Alentejano pigs. Betaine supplementation led to an increase in total cholesterol in both muscles, complementing results previously published indicating a significant increase on the intramuscular lipid content. The expression of twelve genes involved in lipogenesis, lipolysis/FA oxidation, FA transport, and cholesterol metabolism, as well as two transcription factors were also evaluated. Genes related to lipid and cholesterol synthesis plus FA transport were consistently up-regulated in both muscles of betaine fed pigs. On the other hand, genes related to lipolysis/FA oxidation were not affected or down-regulated by betaine supplementation. Our data suggest that the underlying mechanism regulating IMF and cholesterol accumulation in Alentejano pigs supplemented with betaine is associated with the up-regulation of genes involved in lipid synthesis, FA transport, and cholesterol synthesis.

Growth performance, gut health, and metabolism of broilers under thermoneutral and heat stress conditions: multidisciplinary studies on the effects of nutritional strategies and genotype

This PhD project aimed to (i) investigate the effects of three nutritional strategies (supplementation of a synbiotic, a muramidase, or arginine) on growth performance, gut health, and metabolism of broilers fed without antibiotics under thermoneutral and heat stress conditions and to (ii) explore the impacts of heat stress on hypothalamic regulation of feed intake in three broiler lines from diverse stages of genetic selection and in the red jungle fowl, the ancestor of domestic chickens. Synbiotic improved feed efficiency and footpad health, increased Firmicutes and reduced Bacteroidetes in the ceca of birds kept in thermoneutral conditions, while did not mitigate the impacts of heat stress on growth performance. Under optimal thermal conditions, muramidase increased final body weight and reduced cumulative feed intake and feed conversion ratio in a dose-dependent way. The highest dose reduced the risk of footpad lesions, cecal alpha diversity, the Firmicutes to Bacteroidetes ratio, and butyrate producers, increased Bacteroidaceae and Lactobacillaceae, plasmatic levels of bioenergetic metabolites, and reduced the levels of pro-oxidant metabolites. The same dose, however, failed to reduce the effects of heat stress on growth performance. Arginine supplementation improved growth rate, final body weight, and feed efficiency, increased plasmatic levels of arginine and creatine and hepatic levels of creatine and essential amino acids, reduced alpha diversity, Firmicutes, and Proteobacteria (especially Escherichia coli), and increased Bacteroidetes and Lactobacillus salivarius in the ceca of thermoneutral birds. No arginine-mediated attenuation of heat stress was found. Heat stress altered protein metabolism and caused the accumulation of antioxidant and protective molecules in oxidative stress-sensitive tissues. Arginine supplementation, however, may have partially counterbalanced the effects of heat stress on energy homeostasis. Stable gene expression of (an)orexigenic neuropeptides was found in the four chicken populations studied, but responses to hypoxia and heat stress appeared to be related to feed intake regulation.

Ikkε Is Key To Induction Of Insulin Resistance In The Hypothalamus, And Its Inhibition Reverses Obesity.

IKK epsilon (IKKε) is induced by the activation of nuclear factor-κB (NF-κB). Whole-body IKKε knockout mice on a high-fat diet (HFD) were protected from insulin resistance and showed altered energy balance. We demonstrate that IKKε is expressed in neurons and is upregulated in the hypothalamus of obese mice, contributing to insulin and leptin resistance. Blocking IKKε in the hypothalamus of obese mice with CAYMAN10576 or small interfering RNA decreased NF-κB activation in this tissue, relieving the inflammatory environment. Inhibition of IKKε activity, but not TBK1, reduced IRS-1(Ser307) phosphorylation and insulin and leptin resistance by an improvement of the IR/IRS-1/Akt and JAK2/STAT3 pathways in the hypothalamus. These improvements were independent of body weight and food intake. Increased insulin and leptin action/signaling in the hypothalamus may contribute to a decrease in adiposity and hypophagia and an enhancement of energy expenditure accompanied by lower NPY and increased POMC mRNA levels. Improvement of hypothalamic insulin action decreases fasting glycemia, glycemia after pyruvate injection, and PEPCK protein expression in the liver of HFD-fed and db/db mice, suggesting a reduction in hepatic glucose production. We suggest that IKKε may be a key inflammatory mediator in the hypothalamus of obese mice, and its hypothalamic inhibition improves energy and glucose metabolism.

Phosphate Regulated Proteins Of Xanthomonas Citri Subsp. Citri: A Proteomic Approach.

Xanthomonas citri subsp. citri (X. citri) is the causative agent of the citrus canker, a disease that affects several citrus plants in Brazil and across the world. Although many studies have demonstrated the importance of genes for infection and pathogenesis in this bacterium, there are no data related to phosphate uptake and assimilation pathways. To identify the proteins that are involved in the phosphate response, we performed a proteomic analysis of X. citri extracts after growth in three culture media with different phosphate concentrations. Using mass spectrometry and bioinformatics analysis, we showed that X. citri conserved orthologous genes from Pho regulon in Escherichia coli, including the two-component system PhoR/PhoB, ATP binding cassette (ABC transporter) Pst for phosphate uptake, and the alkaline phosphatase PhoA. Analysis performed under phosphate starvation provided evidence of the relevance of the Pst system for phosphate uptake, as well as both periplasmic binding proteins, PhoX and PstS, which were formed in high abundance. The results from this study are the first evidence of the Pho regulon activation in X. citri and bring new insights for studies related to the bacterial metabolism and physiology. Biological significance Using proteomics and bioinformatics analysis we showed for the first time that the phytopathogenic bacterium X. citri conserves a set of proteins that belong to the Pho regulon, which are induced during phosphate starvation. The most relevant in terms of conservation and up-regulation were the periplasmic-binding proteins PstS and PhoX from the ABC transporter PstSBAC for phosphate, the two-component system composed by PhoR/PhoB and the alkaline phosphatase PhoA.

Impaired Incretin Secretion And Pancreatic Dysfunction With Older Age And Diabetes.

To estimate the impact of aging and diabetes on insulin sensitivity, beta-cell function, adipocytokines, and incretin production. Hyperglycemic clamps, arginine tests and meal tolerance tests were performed in 50 non-obese subjects to measure insulin sensitivity (IS) and insulin secretion as well as plasma levels of glucagon, GLP-1 and GIP. Patients with diabetes and healthy control subjects were divided into the following groups: middle-aged type 2 diabetes (MA-DM), aged Type 2 diabetes (A-DM) and middle-aged or aged subjects with normal glucose tolerance (MA-NGT or A-NGT). IS, as determined by the homeostasis model assessment, glucose infusion rate, and oral glucose insulin sensitivity, was reduced in the aged and DM groups compared with MA-NGT, but it was similar in the MA-DM and A-DM groups. Insulinogenic index, first and second phase insulin secretion and the disposition indices, but not insulin response to arginine, were reduced in the aged and DM groups. Postprandial glucagon production was higher in MA-DM compared to MA-NGT. Whereas the GLP-1 production was reduced in A-DM, no differences between groups were observed in GIP production. In non-obese subjects, diabetes and aging impair insulin sensitivity. Insulin production is reduced by aging, and diabetes exacerbates this condition. Aging associated defects superimposed diabetic physiopathology, particularly regarding GLP-1 production. On the other hand, the glucose-independent secretion of insulin was preserved. Knowledge of the complex relationship between aging and diabetes could support the development of physiopathological and pharmacological based therapies.

Up-regulation Of Dnam-1 And Nkp30, Associated With Improvement Of Nk Cells Activation After Long-term Culture Of Mononuclear Cells From Patients With Ovarian Neoplasia.

This study aimed at evaluating the functional activation and activating receptors expression on resting, short- and long-term NK and NK-like T cells from blood of ovarian neoplasia patients. Blood from patients with adnexal benign alterations (n = 10) and ovarian cancer (grade I-IV n = 14) were collected after signed consent. Effector cells activation was evaluated by the expression of the CD107a molecule. Short-term culture was conducted overnight with IL-2 and long-term culture for 21 days, by a method designed to expand CD56(+) lymphocytes. Short-term culture significantly increased NK cells activation compared to resting NK cells (p<0.05), however, the long-term procedure supported an even higher increase (p<0.001). Resting NK-like T cells showed poor activation, which was not altered by the culture procedures. The long-term culture effectively increased the expression of the activating receptors on NK and NK-like T cells, either by increasing the number of cells expressing a given receptor and/or by up-regulating their expression intensity. As a conclusion, the long-term culture system employed, resulted in a high number of functional NK cells. The culture system was particularly efficient on the up-regulation of NKp30 and DNAM-1 receptors on NK cells.

Metabolic Memory Of ß-cells Controls Insulin Secretion And Is Mediated By Camkii.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) functions both in regulation of insulin secretion and neurotransmitter release through common downstream mediators. Therefore, we hypothesized that pancreatic ß-cells acquire and store the information contained in calcium pulses as a form of metabolic memory, just as neurons store cognitive information. To test this hypothesis, we developed a novel paradigm of pulsed exposure of ß-cells to intervals of high glucose, followed by a 24-h consolidation period to eliminate any acute metabolic effects. Strikingly, ß-cells exposed to this high-glucose pulse paradigm exhibited significantly stronger insulin secretion. This metabolic memory was entirely dependent on CaMKII. Metabolic memory was reflected on the protein level by increased expression of proteins involved in glucose sensing and Ca(2+)-dependent vesicle secretion, and by elevated levels of the key ß-cell transcription factor MAFA. In summary, like neurons, human and mouse ß-cells are able to acquire and retrieve information.

Exposure To Bisphenol-a During Pregnancy Partially Mimics The Effects Of A High-fat Diet Altering Glucose Homeostasis And Gene Expression In Adult Male Mice.

Bisphenol-A (BPA) is one of the most widespread EDCs used as a base compound in the manufacture of polycarbonate plastics. The aim of our research has been to study how the exposure to BPA during pregnancy affects weight, glucose homeostasis, pancreatic β-cell function and gene expression in the major peripheral organs that control energy flux: white adipose tissue (WAT), the liver and skeletal muscle, in male offspring 17 and 28 weeks old. Pregnant mice were treated with a subcutaneous injection of 10 µg/kg/day of BPA or a vehicle from day 9 to 16 of pregnancy. One month old offspring were divided into four different groups: vehicle treated mice that ate a normal chow diet (Control group); BPA treated mice that also ate a normal chow diet (BPA); vehicle treated animals that had a high fat diet (HFD) and BPA treated animals that were fed HFD (HFD-BPA). The BPA group started to gain weight at 18 weeks old and caught up to the HFD group before week 28. The BPA group as well as the HFD and HFD-BPA ones presented fasting hyperglycemia, glucose intolerance and high levels of non-esterified fatty acids (NEFA) in plasma compared with the Control one. Glucose stimulated insulin release was disrupted, particularly in the HFD-BPA group. In WAT, the mRNA expression of the genes involved in fatty acid metabolism, Srebpc1, Pparα and Cpt1β was decreased by BPA to the same extent as with the HFD treatment. BPA treatment upregulated Pparγ and Prkaa1 genes in the liver; yet it diminished the expression of Cd36. Hepatic triglyceride levels were increased in all groups compared to control. In conclusion, male offspring from BPA-treated mothers presented symptoms of diabesity. This term refers to a form of diabetes which typically develops in later life and is associated with obesity.

Reduced Insulin Clearance And Lower Insulin-degrading Enzyme Expression In The Liver Might Contribute To The Thrifty Phenotype Of Protein-restricted Mice.

Nutrient restriction during the early stages of life usually leads to alterations in glucose homeostasis, mainly insulin secretion and sensitivity, increasing the risk of metabolic disorders in adulthood. Despite growing evidence regarding the importance of insulin clearance during glucose homeostasis in health and disease, no information exists about this process in malnourished animals. Thus, in the present study, we aimed to determine the effect of a nutrient-restricted diet on insulin clearance using a model in which 30-d-old C57BL/6 mice were exposed to a protein-restricted diet for 14 weeks. After this period, we evaluated many metabolic variables and extracted pancreatic islet, liver, gastrocnemius muscle (GCK) and white adipose tissue samples from the control (normal-protein diet) and restricted (low-protein diet, LP) mice. Insulin concentrations were determined using RIA and protein expression and phosphorylation by Western blot analysis. The LP mice exhibited lower body weight, glycaemia, and insulinaemia, increased glucose tolerance and altered insulin dynamics after the glucose challenge. The improved glucose tolerance could partially be explained by an increase in insulin sensitivity through the phosphorylation of the insulin receptor/protein kinase B and AMP-activated protein kinase/acetyl-CoA carboxylase in the liver, whereas the changes in insulin dynamics could be attributed to reduced insulin secretion coupled with reduced insulin clearance and lower insulin-degrading enzyme (IDE) expression in the liver and GCK. In summary, protein-restricted mice not only produce and secrete less insulin, but also remove and degrade less insulin. This phenomenon has the double benefit of sparing insulin while prolonging and potentiating its effects, probably due to the lower expression of IDE in the liver, possibly with long-term consequences.

Oligomerization, Membrane Association, And In Vivo Phosphorylation Of Sugarcane Udp-glucose Pyrophosphorylase.

Sugarcane is a monocot plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk. The mechanisms that are involved in sucrose accumulation in sugarcane are not well understood, and little is known with regard to factors that control the extent of sucrose storage in the stalks. UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is an enzyme that produces UDP-glucose, a key precursor for sucrose metabolism and cell wall biosynthesis. The objective of this work was to gain insights into the ScUGPase-1 expression pattern and regulatory mechanisms that control protein activity. ScUGPase-1 expression was negatively correlated with the sucrose content in the internodes during development, and only slight differences in the expression patterns were observed between two cultivars that differ in sucrose content. The intracellular localization of ScUGPase-1 indicated partial membrane association of this soluble protein in both the leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo at the Ser-419 site in the soluble and membrane fractions from the leaves but not from the internodes. The purified recombinant enzyme was kinetically characterized in the direction of UDP-glucose formation, and the enzyme activity was affected by redox modification. Preincubation with H2O2 strongly inhibited this activity, which could be reversed by DTT. Small angle x-ray scattering analysis indicated that the dimer interface is located at the C terminus and provided the first structural model of the dimer of sugarcane UGPase in solution.