Incorporation Of 14C-glucose Research Articles

Long-term hypoxia exposure impairs metabolic pathways of de novo lipogenesis in young mouse adipocytes

Introduction: Obstructive sleep apnea syndrome, manifested by tissue hypoxia, is causally linked with the development of metabolic diseases such as Type 2 diabetes. Although there is convincing epidemiological evidence, molecular mechanisms mediating the adverse metabolic effects remain unclear. Hypoxia may play an important role and modulate metabolic pathways in young adipocytes including the utilization of glucose and acetate or the activation of the reverse tricarboxylic acid cycle (rTCA) leading to increase de novo lipogenesis and excessive fat accumulation. The aim of this study was to investigate the impact of long-term hypoxia on important pathways of de novo lipogenesis in mouse early and late adipocytes. Hypothesis: Hypoxia affects the utilization of carbon sources for de novo lipogenesis differently in early and late adipocytes. Methods: Mouse 3T3-L1 cells were differentiated into adipocytes for 7 days - early adipocytes (EA) or 14 days - late adipocytes (LA). The cells were cultured in gas-permeable cultureware allowing exposure to sustained oxygen levels of 21% O 2 or 4% O 2 . [ 13 C-5]-glutamine (2.5mM) was added to culture media for 7 and 14 days to assess 13 C incorporation to newly synthesized lipids via rTCA and [ 13 C-1]-glutamine (2.5mM) was added to media for 24h to assess 13 C incorporation to rTCA metabolites. The 13 C incorporation studies were analyzed via gas chromatography-mass spectrometry. The contribution of glucose and acetate to de novo lipogenesis was assessed using [ 14 C]-glucose and [ 14 C]-acetate (both 0.2 μCi) provided in culture media for 2h at the end of experiments. Subsequently, radioactivity in extracted lipids was measured via scintillation counter. Statistical significance was assessed by 2-way ANOVA followed by Tukey's test. Data are presented as means ± SD, N=9. Data Hypoxia had no effect on lipid accumulation in EA, lipid accumulation in LA was increased by 134,41% vs control. The 13 C incorporation to lipids was increased in EA by 42,2% vs control and by 13,33% vs LA. The 13 C incorporation in citrate was increased by 8,71%, in malate by 9,58% in EA vs control, in LA the incorporation remained unchanged. The [ 14 C]-glucose incorporation to lipids was elevated in EA vs control by 209,98% and EA vs LA by 60,02%. The [ 14 C]-acetate incorporation to lipids was increased in EA by 198,43% vs control and decreased by 45,13% vs LA. Differences are statistically significant (p <0.05). Summary: The effect of hypoxia on lipid accumulation was observed at the late stages of adipocyte maturation. However, in EA hypoxia initially stimulated the activation of rTCA to partially contribute to de novo lipogenesis and significantly increased 14 C-glucose incorporation. In LA the rTCA and 14 C-glucose incorporation were reduced to the control level and the contribution of acetate was increased. Conclusion: Hypoxia modified metabolic pathways in maturating adipocytes leading to increased de novo lipogenesis and thus hypertrophic mature adipocytes. Project was supported by Grant Agency of the Charles University project GAUK 294822 and by grant AZV project NU21-01-00259 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Visceral mesenchymal stem cells from type 2 diabetes donors activate triglycerides synthesis in healthy adipocytes via metabolites exchange and cytokines secretion.

In recent years, there has been an increase in the prevalence of obesity and type 2 diabetes mellitus (T2DM). Development of visceral instead of subcutaneous adipose tissue is pathogenic and increases the risk of metabolic abnormalities. We hypothesize that visceral adipocytes and stromal cells are able to deteriorate other fat depots metabolism via secretory mechanisms. We study the regulatory role of visceral adipose-derived stem cells (vADSC) from donors with obesity and T2DM or normal glucose tolerance (NGT) on healthy subcutaneous ADSC (sADSC) in the Transwell system. Lipid droplets formation during adipogenesis was assessed by confocal microscopy. Cell metabolism was evaluated by 14C-glucose incorporation analysis and western blotting. vADSC secretome was assessed by Milliplex assay. We showed that both NGT and T2DM vADSC had mesenchymal phenotype, but expression of CD29 was enhanced, whereas expressions of CD90, CD140b and IGF1R were suppressed in both NGT and T2DM vADSC. Co-differentiation with T2DM vADSC increased lipid droplet size and stimulated accumulation of fatty acids in adipocytes from healthy sADSC. In mature adipocytes T2DM vADSC stimulated triglyceride formation, whereas NGT vADSC activated oxidative metabolism. Secretome of NGT vADSC was pro-inflammatory and pro-angiogenic in comparison with T2DM vADSC. The present study has demonstrated the critical role of secretory interactions between visceral and subcutaneous fat depots both in the level of progenitor and mature cells. Mechanisms of these interactions are related to direct exchange of metabolites and cytokines secretion.

1328-P: Infant Mesenchymal Stem Cell Insulin Sensitivity Is Associated with Maternal Plasma-Free Fatty Acids, Independent of Obesity Status: The Healthy Start Study

Introduction: Maternal obesity is associated with greater neonatal adiposity and increased offspring risk for type 2 diabetes (T2D) later in life. We have previously reported that mesenchymal stem cells (MSCs) isolated from umbilical cord tissue maintain similar metabolic phenotype to the infant, particularly when differentiated to adipocytes or myocytes. A well-established driver in the pathogenesis of T2D is skeletal muscle insulin resistance. Therefore, we hypothesized that MSCs from infants born to mothers with obesity would display insulin resistance when differentiated to myocytes. Objectives: The objective was to determine the effect of maternal obesity on offspring MSC insulin sensitivity (Si). Methods: MSCs were isolated from umbilical cord tissue from offspring born to either normal weight (BMI 21.1 ± 0.9, n = 9) or obese (BMI 32.9 ± 2.1, n = 10) mothers enrolled in the Healthy Start pre-birth cohort study in Colorado. Following differentiation into skeletal muscle myocytes, MSCs were serum starved and incubated with 14C-glucose, in the presence or absence of insulin. The rate of insulin-mediated 14C-glucose incorporation into glycogen, was used as an index of offspring Si. Results: Offspring Si did not correlate with maternal BMI (r = 0.26), nor was BMI different between groups when median-stratified by offspring Si. However, maternal free fatty acid levels positively correlated with offspring Si (r = 0.63, p < 0.05) and were significantly elevated in the high Si group (low Si vs. high Si; 332.1 ± 30.9 vs. 483.3 ± 48.7 uEq/L, p < 0.05). Conclusions: Offspring Si was not related to the BMI of the mother. Moreover, offspring Si was higher in cells from infants born to mothers with higher free fatty acids during pregnancy. Considering the role of insulin in somatic growth pathways, this phenomenon may serve to increase available nutrient storage capacity during early development. Disclosure A.B. Chaves: None. J.A. Johnson: None. D. Zheng: None. D. Dabelea: None. J.A. Houmard: None. K.E. Boyle: None. Funding American Heart Association (14PRE18230008); Nutrition Obesity Research Center (P30DK048520); National Institutes of Health (R01DK076648); Colorado National Center for Advancing Translational Sciences (UL1TR001082)

Impact of single and binary mixtures of phenanthrene and N-PAHs on microbial utilization of 14C-glucose in soil

Microbes are susceptible to contaminant effects, and high concentration of chemicals in soil can impact on microbial growth, density, viability and development. The impact of single and binary mixtures of phenanthrene and its nitrogen-containing polycyclic aromatic hydrocarbon analogues (N-PAHs) on microbial metabolism of 14C-glucose in soil was measured over a 90 d soil-contact time. Impacts were assessed by measuring the rates and mean overall extents of mineralisation (%), as well as the incorporation of 14C-glucose into the microbial biomass. The result revealed that the extents of 14C-glucose mineralisation were consistently greater in N-PAH amended soils than the control and phenanthrene soils with increased incubations. This indicates a trend of increasing diversion of C from biosynthesis to maintenance requirement by soil microorganisms. Furthermore, biomass uptake in the amended soils showed reduced substrate utilization (fixed-kEC), suggesting that N-PAHs decreased the amount of substrate-C that was incorporated into the microbial biomass. This however, signifies that N-PAHs imposes oxidative stress on soil microbial community.

22 - Vitamin E (RRR-α-tocopherol) decreases hepatic de novo lipogenesis

Non-alcoholic fatty liver disease (NAFLD) is becoming a major hepatic disease in the Western world. The hallmark feature of NAFLD is steatosis, the accumulation of hepatic triacylglycerol (TG), which results from the disruption of metabolic pathways, one of which is de novo lipogenesis (DNL). RRR-α-tocopherol (αT) can reduce steatosis in animals and NAFLD patients through a yet-unknown mechanism. We aimed to identify the mechanism by which αT decreases liver TG and whether its antioxidant (AO) effect is required for this effect. HepG2 cells cultivated under high glucose medium (25 mmol/L) had a 19% increase in lipid accumulation and 2-fold increase in expression of fatty acid synthase (FASN), a key enzyme in DNL, which were reversed by αT to control levels in a dose dependent manner (25-100 µmol/L, 48h). Similarly, 100 µmol/L of αT decreased incorporation of 14C glucose into lipids by 24%. We confirmed this effect in vivo in plasma samples from NAFLD patients (n=21), obtained before and after 4 weeks of αT supplementation (NCT01792115). Liver TG, measured by 1H-magnetic resonance spectroscopy, decreased in 55% of the patients. The lipogenic index, the ratio of C16:0/C18:2n6 in VLDL, reflects hepatic DNL. Patients with reduction in liver TG after 4 weeks of αT also had a significant reduction in the lipogenic index compared to non-responders (p=0.01). To address if αT reduces DNL through its AO function we synthesized methoxy-αT (m-αT), a lipid-soluble αT derivate lacking the AO function and compared it to αT, trolox (water-soluble αT analogue with AO capacity), and probucol (lipid-soluble AO). In our lipogenic HepG2 model, αT, m-αT, trolox and probucol decreased lipid content by max. 18, 0, 0 and 13%, respectively. Thus, our data indicates that αT reduces hepatic DNL in vitro and in vivo through a mechanism that is likely dependent on its antioxidative activity in the lipid compartment.

α 1-acid glycoprotein inhibits lipogenesis in neonatal swine adipose tissue

Serum α1-acid glycoprotein (AGP) is elevated during late gestation and at birth in the pig and rapidly declines postnatally. In contrast, the pig is born with minimal lipid stores in the adipose tissue, but rapidly accumulates lipid during the first week. The present study examined if AGP can affect adipose tissue metabolism in the neonatal pig. Isolated cell cultures or tissue explants were prepared from dorsal subcutaneous adipose tissue of preweaning piglets. Porcine AGP was used at concentrations of 0, 100, 1000 and 5000 ng/ml medium in 24 h incubations. AGP reduced the messenger RNA (mRNA) abundance of the lipogenic enzymes, malic enzyme (ME), fatty acid synthase and acetyl coA carboxylase by at least 40% (P<0.001). The activity of ME and citrate lyase were also reduced by AGP (P<0.05). Glucose oxidation was reduced by treatment with 5000 ng AGP/ml medium (P<0.05). The 14C-glucose incorporation into fatty acids was reduced by ~25% by AGP treatment for 24 h with 1000 ng AGP/ml medium (P<0.05). The decrease in glucose metabolism by AGP appears to function through an inhibition in insulin-mediated glucose oxidation and incorporation into fatty acids. This was supported by the analysis of the mRNA abundance for sterol regulatory element-binding protein (SREBP), carbohydrate regulatory element-binding protein (ChREBP) and insulin receptor substrate 1 (IRS1), which all demonstrated reductions of at least 23% in response to AGP treatment (P<0.05). These data demonstrate an overall suppression of lipogenesis due to AGP inhibition of lipogenic gene expression in vitro, which the metabolic data and SREBP, ChREBP and IRS1 gene expression analysis suggest is through an inhibition in insulin-mediated events. Second, these data suggest that AGP may contribute to limiting lipogenesis within adipose tissue during the perinatal period, as AGP levels are highest for any serum protein at birth.

Impact of carbon nanomaterials on microbial activity in soil

In this study, effects of an increase in concentration of fullerene-C60, single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes (MWCNTs) or fullerene soot (FS) on overall microbial activity was investigated over a 21 d incubation period. Microbial utilisation of 14C-glucose and uptake of 14C-glucose into the microbial biomass was investigated. For CNM-amended soils, greater extents of 14C-glucose mineralisation were found in the C60-amended soils compared to MWCNT-, SWCNT- or FS-amended soils. In addition, the 100 and 1000 mg kg−1 were consistently found to have higher extents of mineralisation in C60, MWCNT, SWCNT or FS-amended soils, respectively. Further, the incorporation of 14C-glucose into the microbial biomass declined slightly with an increase in concentration in the amended soils, but no consistent pattern was observed. As a result, the biophysical quotient (BQ) increased significantly (P < 0.05), as concentrations increased from 1 mg kg−1 to 1000 mg kg−1 in all C60-, MWCNT-, SWCNT- and FS-amended soils. The results obtained from this study showed that the addition to carbon nanomaterials had no profound impacts on the overall microbial activity, and the overall influence of CNMs on soil microbial activity does not reveal a specific pattern in the short term.

Glucose decomposition and its incorporation into soil microbial biomass depending on land use in Mt. Kilimanjaro ecosystems

Land use change can affect terrestrial C stocks, resulting in increased CO2 flux from soil to the atmosphere. In Africa, conversion of natural ecosystems to agricultural lands is the most common land use change. This study investigated the effects of six land use types occurring in Mt. Kilimanjaro ecosystems i.e. (1) lower montane forest (2) grassland and (3) savannah (natural ecosystems) (4) Chagga homegardens (semi-natural ecosystem) and (5) maize fields and (6) coffee plantation (agroecosystems) on microbial biomass carbon (MBC) and dynamics of 14C labelled glucose added into soil. Decomposition of 14C labelled glucose and its incorporation into microbial biomass in surface soils (0–10 and 10–20 cm) were determined. MBC decreased significantly with increased intensity of land use. Mineralization of the 14C labelled glucose occurred in two phases with contrasting rates: 0–10 days (6–18% of 14C d−1) and 15–65 days (<0.1% of 14C d−1). Land use intensification in agroecosystems led to an average increase of glucose decomposition of 14%. The decay rates of the labile C pool in intensively used agricultural lands were up to three times higher compared to natural ecosystems. The incorporation of 14C glucose into microorganisms ranged between 1 and 7% of 14C input in all soils, and was highest in savannah. Agricultural intensification decreased C content in soil through increased mineralization of organic substances and negatively impacted the upper soil layer more compared to the lower one. Based on these results we conclude that semi-natural ecosystems (e.g. Chagga homegardens) are more sustainable in Mt. Kilimanjaro ecosystems compared to intensive agroecosystems.

Supplementation with Fish Oil as a Source of n–3 Fatty Acids Does Not Downregulate Mammary Lipogenesis in Lactating Mice

The very long chain n–3 (ω-3) polyunsaturated fatty acids (VLCn3PUFAs) are potent regulators of hepatic lipid synthesis, but their effect on lipid synthesis in the lactating mammary gland is less well investigated. The objective of the present study was to examine effects of fish oil (FO) supplementation on mammary lipogenesis and the expression of lipogenic genes in mammary and hepatic tissues of lactating mice. Beginning on day 6 of lactation and continuing for 7 d, female C57BL/6J mice (n = 8/diet) were fed 1 of 3 dietary treatments: a 5%-fat diet containing mainly saturated fatty acids (FAs) (low-fat control) or 2 10%-fat diets, 1 enriched with FO as a source of VLCn3PUFAs and the other enriched with a safflower/palm oil mixture (high-fat control) as a source of oleic acid. Mammary lipogenic capacity, measured by 14C-glucose incorporation into FAs by mammary explants, was similar among treatments, and there were no treatment effects on the proportion of de novo synthesized FAs in milk fat or on litter weight gain, a proxy for milk energy secretion. Also, there were no treatment effects on mammary mRNA abundance for key lipogenic enzymes and proteins involved in the regulation of milk lipid synthesis. In contrast, there was a treatment effect on hepatic lipogenesis, with FO resulting in a decrease of ∼50% in hepatic lipid content and a similar downregulation of lipogenic gene expression compared with the 2 control diets. Overall, there were tissue-specific differences in dietary VLCn3PUFA effects on lipid synthesis with no observed effects for mammary lipogenic variables but marked reductions occurring in hepatic lipogenesis.

Agonism and Antagonism at the Insulin Receptor

Insulin can trigger metabolic as well as mitogenic effects, the latter being pharmaceutically undesirable. An understanding of the structure/function relationships between insulin receptor (IR) binding and mitogenic/metabolic signalling would greatly facilitate the preclinical development of new insulin analogues. The occurrence of ligand agonism and antagonism is well described for G protein-coupled receptors (GPCRs) and other receptors but in general, with the exception of antibodies, not for receptor tyrosine kinases (RTKs). In the case of the IR, no natural ligand or insulin analogue has been shown to exhibit antagonistic properties, with the exception of a crosslinked insulin dimer (B29-B’29). However, synthetic monomeric or dimeric peptides targeting sites 1 or 2 of the IR were shown to be either agonists or antagonists. We found here that the S961 peptide, previously described to be an IR antagonist, exhibited partial agonistic effects in the 1–10 nM range, showing altogether a bell-shaped dose-response curve. Intriguingly, the agonistic effects of S961 were seen only on mitogenic endpoints (3H-thymidine incorporation), and not on metabolic endpoints (14C-glucose incorporation in adipocytes and muscle cells). The agonistic effects of S961 were observed in 3 independent cell lines, with complete concordance between mitogenicity (3H-thymidine incorporation) and phosphorylation of the IR and Akt. Together with the B29-B’29 crosslinked dimer, S961 is a rare example of a mixed agonist/antagonist for the human IR. A plausible mechanistic explanation based on the bivalent crosslinking model of IR activation is proposed.

Butyrate supplementation to gestating sows and piglets induces muscle and adipose tissue oxidative genes and improves growth performance

Weaned pigs often experience growth reduction immediately after weaning due to multiple stress factors associated with weaning. We tested the effect of prenatal and postnatal butyrate supplementation on growth performance of piglets. In study 1, piglets were orally gavaged with 0.3% butyrate from day 4 after birth to weaning (day 21). Butyrate increased ADG by 13% compared to saline treated control. Expression of peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC-1α) was higher in muscle, adipose tissue, and ileum of butyrate-supplemented animals. Also, peroxisome proliferator activated receptor alpha (PPARα) was induced (P < 0.05) in the subcutaneous adipose tissue (SCAT) and muscle (longissimus dorsi [LD]) of butyrate-supplemented piglets. In vitro, butyrate increased (P < 0.05) fatty acid oxidation in primary adipocytes and suppressed basal lipolysis by 62% compared to untreated cells. Butyrate suppressed (P < 0.05) lipogenesis ((14)C-glucose incorporation into lipids) in adipocytes. This was accompanied by an approximately 30% reduction in the mRNA expression of fatty acid synthase (P < 0.05) in butyrate-treated cells vs. controls. Piglets born to sows that were supplemented with 0.3% butyrate during the last trimester of gestation had a 15% higher (P < 0.05) body weight at 12 wk than controls. In summary, butyrate supplementation to gestating sows and piglets enhanced postweaning growth performance, which may be mediated by increased substrate oxidation in butyrate treated animals.

Rapid Turnover of Glycogen in Memory Formation

The influence of noradrenaline acting at α(2)-AR and β(2)-ARs on the turnover of glycogen after learning has been investigated. The role of glycogen turnover in memory formation was examined using weakly-reinforced, single trial bead discrimination training in day-old domestic chickens. This study follows our previous work that focused on the need for glycogen breakdown (glycogenolysis) during learning. Inhibition of glycogenolysis by 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) prevented the consolidation of strongly-reinforced learning and inhibited memory. The action of DAB could be prevented by stimulating glycogenolysis with the selective β(2)-AR agonist, zinterol. Stimulation of α(2)-ARs has been shown to lead to an increase in the turnover and synthesis of glycogen. In the present study, we examined the effect of inhibition of α(2)-AR stimulated glycogen turnover (measured as(14)C-glucose incorporation into glycogen) on the ability of zinterol to promote the consolidation of weakly reinforced memory. In astrocytes, the selective α(2)-AR agonist clonidine stimulated (14)C-glucose incorporation into glycogen in chick astrocytes and this was inhibited by the selective α(2)-AR antagonist, ARC239. The critical importance of the timing of ARC239 injection relative to training and intracerebral administration of zinterol was examined. It is concluded that our data provides evidence for a readily accessible labile pool of glycogen in brain astrocytes. If glycogen synthesis is inhibited, the can be depleted within 10 min, thus preventing zinterol from promoting consolidation.

Valine Metabolism of Mongolian Gerbil (Meriones Unguiculatus) Embryos

Abstract: Mongolian gerbil 1-cell embryos do not develop into blastocysts in vitro because of 8-cell block. Recently, we reported that 2-cell embryos of Mongolian gerbils developed into blastocysts in vitro in mM16 supplemented with valine. However, the role of valine in the in vitro development of Mongolian gerbil embryos is unclear. Thus, the objective of this study was to investigate of the role of valine in the in vitro development of Mongolian gerbil embryos. Mongolian gerbil embryos at the 1-cell, 2-cell, 8-cell, morula, and blastocyst stages were cultured in mM16 medium containing 14C-valine or 14C-glucose. A 2.5 mM NaOH solution was used as a trap for the evolved 14CO2. After incubation, all samples were analyzed using a liquid scintillation counter. The incorporation of 14C-valine significantly increased at the 8-cell stage. The oxidation of 14C-valine significantly increased at the 8-cell and blastocyst stages. The incorporation of 14C-glucose in mM16+valine was significantly higher than that in...

Role of fatty acid elongases in determination of de novo synthesized monounsaturated fatty acid species

Enhanced production of monounsaturated fatty acids (FA) derived from carbohydrate-enriched diets has been implicated in the development of obesity and insulin resistance. The FA elongases Elovl-5 and Elovl-6 are regulated by nutrient and hormone status, and have been shown using intact yeast and mammalian microsome fractions to be involved in the synthesis of monounsaturated FAs (MUFA). Herein, targeted knockdown and overexpression of Elovl-5 or Elovl-6 was used to determine their roles in de novo synthesis of specific MUFA species in mammalian cells. Treatment of rat insulinoma (INS)-1 cells with elevated glucose increased de novo FA synthesis and the ratio of MUFAs to saturated FAs. Elovl-5 knockdown decreased elongation of 16:1,n-7. Elovl-5 overexpression increased synthesis of 18:1,n-7; however, this increase was dependent on stearoyl-CoA desaturase-driven 16:1,n-7 availability. Knockdown of Elovl-6 decreased elongation of 16:0 and 16:1,n-7, resulting in accumulation of 16:1,n-7. Elovl-6 overexpression preferentially drove synthesis of 16:0 elongation products 18:0 and 18:1,n-9 but not 18:1,n-7. These findings demonstrate that coordinated induction of FA elongase and desaturase activity is required for balanced synthesis of specific n-7 versus n-9 MUFA species. Given the relative abundance of 16:0 to 16:1,n-7 and the specificity of Elovl-6 for 16:0, Elovl-6 is a major elongase for 18:1,n-9 production.

The beneficial effect of fructose and glucose on in vitro maturation and the fertilization of porcine oocytes.

This study was conducted to investigate the effects of supplementing fructose in the culture medium on in vitro maturation (IVM), in vitro fertilization (IVF), and metabolism of porcine oocytes. Porcine oocytes were matured in vitro in modified North Carolina State University 37 medium (NCSU-37) and then supplemented with either glucose (5.5 mM), fructose (5.5 mM), or glucose (2.75 mM) plus fructose (2.75 mM). The maturation and fertilization of oocytes, and the incorporation and oxidation of 14C-glucose, 14C-fructose, and 14C-methionine in oocytes at different stages of development were examined. The supplementation of glucose plus fructose significantly promoted (P < 0.05) oocytes germinal vesicle break down (GVBD), maturation to metaphase II (MII), penetration by spermatozoa, and male pronuclear formation compared with glucose. The incorporation and oxidation of 14C-methionine into the oocyte significantly increased (P < 0.05) with glucose plus fructose supplementation than glucose. A significantly higher (P < 0.05) rate of incorporation and oxidation was achieved with 14C-fructose compared to 14C-glucose. Glucose plus fructose supplementation improved maturation, penetration by spermatozoa, male pronuclear formation, and energy metabolism by porcine oocytes.

Nitric oxide: an active nitrogen molecule that modulates cellulose synthesis in tomato roots.

Nitric oxide (NO) is a bioactive molecule involved in several growth and developmental processes in plants. These processes are mostly characterized by changes in primary and secondary metabolism. Here, the effect of NO on cellulose synthesis in tomato (Solanum lycopersicum) roots was studied. The phenotype of roots, cellulose content, the incorporation of 14C-glucose into cellulosic fraction and the expression of tomato cellulose synthase (CESA) transcripts in roots treated with the NO donor sodium nitroprusside (SNP) were analysed. Nitric oxide affected cellulose content in roots in a dose dependent manner. Low concentrations of SNP (pmoles of NO) increased cellulose content in roots while higher concentrations of SNP (nmoles of NO) had the opposite effect. This result correlated with assays of 14C-glucose incorporation into cellulose in roots. The effect of NO on 14C-glucose incorporation into cellulose was transient and reversible. Microscopic analysis of roots suggested that NO affected primary cell wall cellulose synthesis. Three tomato cellulose synthase (SICESA) transcripts were identified. Reverse transcriptase polymerase chain reaction experiments were carried out and indicated that SICESA1 and SICESA3 levels were affected by high NO concentrations. Together, these results support the hypothesis that variations in NO levels influence cellulose synthesis and content in roots.

Effect of fatty acids bound to bovine serum albumin-V on acrosome reaction and utilization of glucose in boar spermatozoa.

Aim: The present study has been designed with the objective of determining if fatty acids bound to bovine serum albumin-V (BSA-V) can improve motility, viability, and increase acrosome reaction (AR) and utilization of glucose in boar spermatozoa. Methods: Boar spermatozoa were washed, swum-up and incubated at 37°C for 6h in TALP medium supplemented with fatty acids bound to bovine serum albumin fraction V (BSA-V), fatty acid free BSA (BSA-FAF), polyvinyl alcohol+ main fatty acids bound to BSA-V (PVA+FA) and PVA. Sperm motility, viability, AR, and the incorporation and oxidation of 14C-glucose were evaluated during 6h of incubation. Results: The results show that the BSA-V was superior to BSA-FAF and PVA in improving motility and AR. Viability was significantly increased (P<0.05) by only BSA-V compared with PVA. When the main fatty acids compound of BSA-V were added to PVA, the sperm motility, viability and AR became almost the same as with BSA-V. The rate of incorporation and oxidation of 14C-glucose were significantly increased (P<0.05) by BSA-V compared with BSA-FAF and PVA. Fatty acids bound to BSA-V are important for improvement of sperm functions. Conclusions: The present study postulates that fatty acids bound to BSA-V are important to acrosome reaction and the utilization of glucose in boar spermatozoa.

Uptake of 14C-glucose by Botryodiplodia theobromae Pat and changes in its carbohydrate content during growth

There is a rapid uptake of 14C-glucose and unlabelled glucose during the period of pigmentation of the mycelium of Botryodiplodia theobromae Pat. Incorporation of 14C-glucose into structural components of the mycelium started within 24 hours of incubation and progressively increased until the 14th day of growth. The mycelium contains glucose, isomaltose, and sucrose. There is a decrease in these carbohydrates during autolysis. Autolysis in B. theobromae occurs in an acid medium of pH 5.2.

The use of antibiotics to reduce bacterioplankton uptake of phytoplankton extracellular organic carbon (EOC) in the Potomac River estuary

Phytoplankton production and accumulation of extracellular organic carbon (EOC) was tracked during diel intervals in microcosms by inhibiting bacterioplankton assimilation of EOC with streptomycin and kanamycin. Bacterioplankton production ( 3H-thymidine incorporation) and metabolism ( 14C-glucose incorporation) were monitored in samples collected from the Potomac River estuary to determine the effect of the antibiotics. Particulate (i.e., raw water) primary production and EOC (i.e., water passing through 1.0 μm glass fiber filter) production rates were monitored to determine the impact of antibiotics on phytoplankton. In preliminary experiments, neither streptomycin nor kanamycin alone significantly inhibited bacterioplankton activity compared to controls, but when both were present secondary production and metabolism were reduced up to 90%, and remained as such for 45 h. During field evaluations using a streptomycin and kanamycin mixture (50 μM each) particulate primary production and EOC production were not statistically different in control and antibiotic treated samples indicating that the antibiotics did not negatively influence phytoplankton production rates. In the presence of antibiotics dissolved free amino acids (DFAA) and, to a lesser extent, monosaccharides were significantly elevated compared to controls. This study demonstrates that streptomycin and kanamycin are capable of inhibiting bacterioplankton metabolism and uptake of dissolved organic carbon (DOC) in the samples tested so that the contribution of EOC to the DOC pool and to bacterioplankton metabolism could be measured and assessed.

Effect of relaxin on motility, acrosome reaction and viability of cryopreserved boar spermatozoa.

Background and Aims: Relaxin has an important role in stimulating motility and the acrosome reaction (AR) of fresh boar spermatozoa. The objective of the present study was to determine whether relaxin can improve the motility, AR and viability of cryopreserved boar spermatozoa. Methods: Cryopreserved boar spermatozoa were thawed, washed and incubated at 37°C for 4h in modified Beltsville thawing solution supplemented with 0, 20 or 40ng/mL relaxin. Sperm motility, AR, viability, and incorporation and oxidation of 14C-glucose were evaluated during 0-4h of incubation. Results: The results show that the supplementation of relaxin (especially at 20ng/mL) in the thawing solution improved sperm motility significantly (P<0.05) at 1-3h of incubation. The percentage of acrosome reacted live spermatozoa was improved significantly (P<0.05) when the spermatozoa were treated with 20ng/mL relaxin. Viability was not significantly (P>0.05) improved by supplementation with relaxin. The rates of incorporation and oxidation of 14C-glucose were increased in correlation with AR up to 4h of incubation. Conclusion: We conclude that relaxin can improve the sperm motility and AR, and enhance the glucose metabolism of cryopreserved boar spermatozoa. (Reprod Med Biol 2006; 5: 215-220).