Incorporation Of Radiolabeled Glucose Research Articles

Discovery and Characterization of Fluopipamine, a Putative Cellulose Synthase 1 Antagonist within Arabidopsis.

Herbicide-resistant weeds are increasingly a problem in crop fields when exposed to similar chemistry over time. To avoid future yield losses, identifying herbicidal chemistry needs to be accelerated. We screened 50,000 small molecules using a liquid-handling robot and light microscopy focusing on pre-emergent herbicides in the family of cellulose biosynthesis inhibitors. Through phenotypic, chemical, genetic, and in silico methods we uncovered 6-{[4-(2-fluorophenyl)-1-piperazinyl]methyl}-N-(2-methoxy-5-methylphenyl)-1,3,5-triazine-2,4-diamine (fluopipamine). Symptomologies support fluopipamine as a putative antagonist of cellulose synthase enzyme 1 (CESA1) from Arabidopsis (Arabidopsis thaliana). Ectopic lignification, inhibition of etiolation, phenotypes including loss of anisotropic cellular expansion, swollen roots, and live cell imaging link fluopipamine to cellulose biosynthesis inhibition. Radiolabeled glucose incorporation of cellulose decreased in short-duration experiments when seedlings were incubated in fluopipamine. To elucidate the mechanism, ethylmethanesulfonate mutagenized M2 seedlings were screened for fluopipamine resistance. Two loci of genetic resistance were linked to CESA1. In silico docking of fluopipamine, quinoxyphen, and flupoxam against various CESA1 mutations suggests that an alternative binding site at the interface between CESA proteins is necessary to preserve cellulose polymerization in compound presence. These data uncovered potential fundamental mechanisms of cellulose biosynthesis in plants along with feasible leads for herbicidal uses.

FAM13A and POM121C are candidate genes for fasting insulin: functional follow-up analysis of a genome-wide association study

Aims/hypothesisBy genome-wide association meta-analysis, 17 genetic loci associated with fasting serum insulin (FSI), a marker of systemic insulin resistance, have been identified. To define potential culprit genes in these loci, in a cross-sectional study we analysed white adipose tissue (WAT) expression of 120 genes in these loci in relation to systemic and adipose tissue variables, and functionally evaluated genes demonstrating genotype-specific expression in WAT (eQTLs).MethodsAbdominal subcutaneous adipose tissue biopsies were obtained from 114 women. Basal lipolytic activity was measured as glycerol release from adipose tissue explants. Adipocytes were isolated and insulin-stimulated incorporation of radiolabelled glucose into lipids was used to quantify adipocyte insulin sensitivity. Small interfering RNA-mediated knockout in human mesenchymal stem cells was used for functional evaluation of genes.ResultsAdipose expression of 48 of the studied candidate genes associated significantly with FSI, whereas expression of 24, 17 and 2 genes, respectively, associated with adipocyte insulin sensitivity, lipolysis and/or WAT morphology (i.e. fat cell size relative to total body fat mass). Four genetic loci contained eQTLs. In one chromosome 4 locus (rs3822072), the FSI-increasing allele associated with lower FAM13A expression and FAM13A expression associated with a beneficial metabolic profile including decreased WAT lipolysis (regression coefficient, R = −0.50, p = 5.6 × 10−7). Knockdown of FAM13A increased lipolysis by ~1.5-fold and the expression of LIPE (encoding hormone-sensitive lipase, a rate-limiting enzyme in lipolysis). At the chromosome 7 locus (rs1167800), the FSI-increasing allele associated with lower POM121C expression. Consistent with an insulin-sensitising function, POM121C expression associated with systemic insulin sensitivity (R = −0.22, p = 2.0 × 10−2), adipocyte insulin sensitivity (R = 0.28, p = 3.4 × 10−3) and adipose hyperplasia (R = −0.29, p = 2.6 × 10−2). POM121C knockdown decreased expression of all adipocyte-specific markers by 25–50%, suggesting that POM121C is necessary for adipogenesis.Conclusions/interpretationGene expression and adipocyte functional studies support the notion that FAM13A and POM121C control adipocyte lipolysis and adipogenesis, respectively, and might thereby be involved in genetic control of systemic insulin sensitivity.

Family history of diabetes is associated with enhanced adipose lipolysis: Evidence for the implication of epigenetic factors

AimsType 2 diabetes is associated with insulin resistance, adipose hypertrophy and increased lipolysis. The heritability of these traits has been determined by associating them with a family history of diabetes. MethodsAbdominal subcutaneous fat biopsies were obtained from 581 subjects in a cross-sectional study. Fat cells were isolated, and the difference between measured and expected fat-cell volume was used to determine adipose morphology (degree of hypertrophy or hyperplasia). Spontaneous lipolytic activity was determined in explants of adipose tissue by measuring glycerol release. Insulin-stimulated lipogenesis was assessed by measuring the incorporation of radiolabelled glucose into fat-cell lipids. Information on parental history of diabetes was gathered by a questionnaire. ResultsAdipose morphology correlated positively with lipolysis (P<0.0001) and inversely with insulin-stimulated lipogenesis (P<0.008). Also, 24% of probands had a family history of diabetes, which was associated with higher body mass index (BMI) scores, and more insulin resistance (HOMAIR) and adipose hypertrophy. Lipolytic activity was increased, and insulin-stimulated lipogenesis decreased, in probands with a parental history of diabetes. The results for HOMAIR, lipolysis and adipose morphology remained significant after adjusting for proband BMI. A maternal history of diabetes was associated with increased adipose lipolytic activity in probands. ConclusionA family history of diabetes is independent of proband BMI, but associated with adipocyte hypertrophy and enhanced lipolysis, which suggests that these factors are genetically linked to diabetes. Moreover, the influence on lipolysis was only observed in probands with a maternal history of diabetes, thereby supporting an epigenetic impact.

Stress of endoplasmic reticulum modulates differentiation and lipogenesis of human adipocytes

BackgroundAdipocytes are cells specialized for storage of neutral lipids. This storage capacity is dependent on lipogenesis and is diminished in obesity. The reason for the decline in lipogenic activity of adipocytes in obesity remains unknown. Recent data show that lipogenesis in liver is regulated by pathways initiated by endoplasmic reticulum stress (ERS). Thus, we aimed at investigating the effect of ERS on lipogenesis in adipose cells. MethodsPreadipocytes were isolated from subcutaneous abdominal adipose tissue from obese volunteers and in vitro differentiated into adipocytes. ERS was induced pharmacologically by thapsigargin (TG) or tunicamycin (TM). Activation of Unfolded Protein Response pathway (UPR) was monitored on the level of eIF2α phosphorylation and mRNA expression of downstream targets of UPR sensors. Adipogenic and lipogenic capacity was evaluated by Oil Red O staining, measurement of incorporation of radio-labelled glucose or acetic acid into lipids and mRNA analysis of adipogenic/lipogenic markers. ResultsExposition of adipocytes to high doses of TG (100 nM) and TM (1 μg/ml) for 1–24 h enhanced expression of several UPR markers (HSPA5, EDEM1, ATF4, XBP1s) and phosphorylation of eIF2α. This acute ERS substantially inhibited expression of lipogenic genes (DGAT2, FASN, SCD1) and glucose incorporation into lipids. Moreover, chronic exposure of preadipocytes to low dose of TG (2.5 nM) during the early phases of adipogenic conversion of preadipocytes impaired both, lipogenesis and adipogenesis. On the other hand, chronic low ERS had no apparent effect on lipogenesis in mature adipocytes. ConclusionsAcute ERS weakened a capacity of mature adipocytes to store lipids and chronic ERS diminished adipogenic potential of preadipocytes.

Decreased excretion of antimicrobial proteins and peptides in saliva of patients with oral candidiasis

Antimicrobial peptides in saliva appear to play a crucial role in the regulation of oral Candida growth, and study on antimicrobial excretion in saliva and oral candidiasis appears useful for the analysis of pathophysiology of oral candidiasis. To clarify the role of saliva in the regulation of oral Candida growth, the levels of antimicrobial proteins and peptides and their excretion rates were examined in saliva obtained from 50 patients with oral candidiasis and 35 healthy individuals. The inhibitory activities of patients' saliva against Candida adhesion with HeLa cells and against Candida growth (radiolabeled glucose incorporation) were lower than those of saliva from the healthy controls. The salivary levels of lactoferrin (Lf; 11 +/- 9 microg/ml), secretory immunoglobulin A (sIgA; 160 +/- 37 microg/ml), beta-defensin 1 (375 +/- 37 ng/ml), and beta-defensin 2 (412 +/- 51 ng/ml) in the patients were largely lower than those in the control group (33 +/- 14 microg/ml, 204 +/- 51 microg/ml, 452 +/- 89 ng/ml, and 530 +/- 142 ng/ml, respectively), although the transferrin (Tf) and secretory component (SC) levels were almost same in both groups, and alpha-defensin 1 was slightly increased in the patient group (660 +/- 115 ng/ml vs. 467 +/- 168 ng/ml). In addition, the excretion rates of the proteins and peptides were largely decreased in the patients (Tf: 14 +/- 2 microg/10 min vs. 34 +/- 7 microg/10 min; Lf: 18 +/- 11 microg/10 min vs. 139 +/- 43 microg/10 min; sIgA: 300 +/- 132 microg/10 min vs. 900 +/- 207 microg/10 min; SC: 112 +/- 46 microg/10 min vs. 292 +/- 64 microg/10 min; alpha-defensin 1: 1223 +/- 431 ng/10 min vs. 2044 +/- 612 ng/10 min; beta-defensin 1: 687 +/- 243 ng/10 min vs. 1985 +/- 295 ng/10 min; and beta-defensin 2: 784 +/- 299 ng/10 min vs. 2288 +/- 278 ng/10 min). These results conclusively suggest that oral candidiasis is associated with salivary gland hypofunction and that decreases of salivary antibacterial proteins induce Candida overgrowth.

Antibacterial property of isoflavonoids isolated from Erythrina variegata against cariogenic oral bacteria.

The antibacterial property of 7 compounds, isolated from Erythrina variegata (Leguminosae) by repeated silica gel column chromatography, against cariogenic oral bacteria was investigated. Extensive spectroscopic study revealed that all were isoflavonoids. Among them, 3,9-dihydroxy-2,10-di(gamma,gamma-dimethylallyl)-6a,11a-dehydropterocarpan (erycristagallin) showed the highest antibacterial activity against mutans streptococci, other oral streptococci, Actinomyces and Lactobacillus species with a minimum inhibitory concentration (MIC) range of 1.56-6.25 microg/ml, followed by 3,6a-dihydroxy-9-methoxy-2,10-di(gamma,gamma-dimethylallyl)pterocarpan (erystagallinA) and 9-hydroxy-3-methoxy-2-gamma,gamma-dimethylallylpterocarpan (orientanol B) (MIC range: 3.13-12.5 microg/ml). The antibacterial effect of erycristagallin to mutans streptococci was based on a bactericidal action. Erycristagallin (6.25 microg/ml: MIC) completely inhibited incorporation of radio-labelled thymidine into Streptococcus mutans cells. Incorporation of radio-labelled glucose into bacterial cells was also strongly inhibited at MIC, and 1/2 MIC of the compound reduced the incorporation approximately by half. The findings indicate that erycristagallin has a potential as potent phytochemical agent for prevention of dental caries by inhibiting the growth of cariogenic bacteria and by interfering with incorporation of glucose responsible for production of organic acids.

Conjugated Linoleic Acid Inhibits Proliferation but Stimulates Lipid Filling of Murine 3T3-L1 Preadipocytes

This study documented the effects of conjugated linoleic acid (CLA) on the proliferation and differentiation of 3T3-L1 preadipocytes. During proliferation, preadipocytes were cultured in Dulbecco's modified Eagle's medium (DMEM), 100 g/L fetal bovine serum (FBS), 0.584 g/L L-glutamine and 0 (control), 0.5, 1.0, 5.0 or 10.0 mg/L CLA. Proliferation of 3T3-L1 preadipocytes was measured directly by cell counting and indirectly by radiolabeled thymidine incorporation into DNA at 96 h postinoculation. Conjugated linoleic acid was not cytotoxic during proliferation or differentiation. The 0.5, 1.0, 5.0 or 10.0 mg/L CLA treatments inhibited proliferation by 8, 12, 31 and 36%, respectively (all P < 0.05). Treatment with 10 mg/L CLA or 10 mg/L linoleic acid (cis-9,12) reduced the incorporation of 3H-thymidine into DNA by 56 and 35%, respectively, suggesting that some portion of the effect of CLA on preadipocyte proliferation was nonspecific. After the initiation of differentiation, preadipocytes were cultured in DMEM, 100 g/L FBS, 0.584 g/L L-glutamine, 1.7 μmol/L insulin and 0 (control), 0.5, 1.0, 5.0 or 10.0 mg/L CLA. Radiolabeled glucose incorporation into cellular lipids was increased from 7.4 to 11.1, 11.1, 17.4 and 22.5 nmol/(h·106 cells) (all P < 0.05) by 0.5, 1.0, 5.0 and 10.0 mg/L CLA, respectively. A media concentration of 10 mg/L CLA increased total cellular CLA (from 0 to 0.16 ± 0.01 μmol/106 cells), palmitic acid (from 0.47 to 1.10 ± 0.03 μmol/106 cells) and palmitoleic acid (from 0.24 to 0.81 ± 0.03 μmol/106 cells) (means ± pooled SEM; all P < 0.05). Conjugated linoleic acid had no effect on arachidonic acid content, but decreased its proportion (g arachidonic acid/100 g total fatty acids) by >50% (P < 0.05). These data indicate that CLA inhibited proliferation and promoted de novo lipogenesis and lipid filling in 3T3-L1 preadipocytes, suggesting that CLA may reduce overall fat accumulation in growing animals by inhibiting stromal vascular preadipocyte hyperplasia.

Effect of growth hormone treatment on insulin action in adipocytes from children with Prader-Willi syndrome.

To study the effect of growth hormone (GH) treatment (2-4 months) on insulin action in adipocytes isolated from children with Prader-Willi syndrome (PWS), in whom GH deficiency appears to be a primary defect. We investigated the complex effects of GH on carbohydrate metabolism, as part of a current clinical trial of GH treatment in children with PWS. Biopsies of subcutaneous abdominal adipose tissue were obtained from 12 children with PWS before and after 2-4 months of GH treatment. Lipogenesis was determined by the incorporation of radiolabelled glucose into lipids in isolated adipocytes, and glycerol release to the incubation medium was used as an index of lipolysis. GLUT4 RNA was measured by solution hybridization. With low glucose concentrations, at which glucose transport is rate-limiting, maximal insulin-induced lipogenesis was increased by 120% after GH treatment (P < 0.05), but the sensitivity to insulin (half-maximum effective hormone concentration) was unchanged. This was not accompanied by a significant change in the RNA expression of GLUT4. Neither responsiveness (maximum effect) nor sensitivity of insulin-induced inhibition of lipolysis was affected by GH treatment. GH treatment of children with PWS results in an upregulation of insulin-induced lipogenesis in isolated adipocytes, with no effect on insulin-induced inhibition of lipolysis. The data suggest that the site of the effect of GH on lipogenesis is distal to the insulin hormone-receptor interaction, but does not involve altered GLUT4 expression.

Action of PBAN and related peptides on pheromone biosynthesis in isolated pheromone glands of the redbanded leafroller moth, Argyrotaenia velutinana

Isolated pheromone glands from the redbanded leafroller moth, Argyrotaenia velutinana, were utilized to demonstrate the action of pheromone biosynthesis activating neuropeptide (PBAN) and bursa pheromonotropic peptide plus several other related peptides on pheromone biosynthesis. All peptides belonging to the PBAN family and the bursa peptide stimulated pheromone biosynthesis as measured by pheromone titer and incorporation of radiolabeled acetate. These peptides required the presence of extracellular Ca 2+ for expression of full activity and several inorganic Ca 2+ channel blockers inhibited the stimulation of pheromone biosynthesis. The Ca 2+ ionophore A23187 alone stimulated pheromone biosynthesis as did a cAMP analogue. Stimulation by the cAMP analogue in the absence of extracellular Ca 2+ was observed. Maximum pheromone titers were observed in 16 hr gland incubations; however, 2–6 hr incubations were required if pheromone biosynthesis was measured by the incorporation of radiolabeled acetate. Radiolabeled glucose incorporation was not increased in the presence of PBAN. These results are discussed in the context of how the pheromone biosynthetic pathway is stimulated by these peptides.

Glucose Uptake and End Product Formation in an Intertidal Marine Sediment

Glucose uptake was monitored on a seasonal basis, using [6-H]glucose and undisturbed cores collected from an intertidal mud flat. The fate of glucose carbon, including the formation of CO(2) and biomass, was assayed by using undisturbed cores and [U-C]glucose; the production of short-chain fatty acids was monitored with [U-C]glucose and sediment slurries. Rate constants for glucose uptake varied temporally, with temperature accounting for much of the variability; turnover times ranged from about 2 to 10 min. Rate constants decreased with increasing sediment depth and in the following order for several common monosaccharides: glucose>galactose>mannose approximately fucose. Time course analyses of CO(2) production provided evidence of significant isotopic dilution; although pore water glucose turnover times were on the order of minutes, CO(2) did not plateau until after approximately 6 h of incubation. At this time a maximum of about 40% of the added radioglucose had been respired. The extent of respiration varied as a function of sediment depth and season, with the highest values below the surface (4 to 7 cm) and in summer and fall. Incorporation of radiolabelled glucose into biomass also varied seasonally, but the greatest extent of incorporation (about 40%) was observed in the fall and for the 0- to 1-cm depth interval. The production of short-chain fatty acid end products was largely limited to acetate, which accounted for only a small percentage of the added radiolabel. Other organic acids, pyruvate in particular, were observed in pore water and were due to artifacts in the heat-kill procedure used to terminate incubations. An accurate assessment of the distribution and importance of short-chain fatty acids as end products required the use of an enzymatic technique coupled with high-pressure liquid chromatography to verify qualitative identities.

Lysozyme enhances the inhibitory effects of the peroxidase system on glucose metabolism of Streptococcus mutans.

The combined effect of the salivary peroxidase system and lysozyme on the glucose uptake of Streptococcus mutans NCTC 10449 was investigated. The bacteria were grown to late-exponential phase, washed, re-suspended in buffer at pH6, and incubated with (1) 50 micrograms/mL lysozyme from human milk for 60 min; (2) 7-15 mumol/L hypothiocyanous acid/hypothiocyanite for 10 min; and (3) lysozyme for 60 min prior to addition of and incubation with hypothiocyanous acid/hypothiocyanite for 10 min. Glucose uptake was initiated by adding the bacterial suspensions to 10 mL of pre-warmed 50 mumol/L glucose containing 0.98 mumol/L D-(U-14C-)-glucose, and the mixture was incubated in a shaking water-bath at 37 degrees C. Samples were withdrawn at various time intervals, rapidly filtered through 0.45-microns membranes, washed with ice-chilled buffer, and the incorporated radioactivity determined. Lysozyme stimulated S. mutans glucose uptake slightly, but significantly inhibited S. rattus glucose metabolism. A 20-30% inhibition of radiolabeled glucose incorporation was observed with hypothiocyanous acid/hypothiocyanite alone. Incubation of the bacteria with lysozyme prior to addition of hypothiocyanous acid/hypothiocyanite containing peroxidase resulted in a total inhibition of the glucose uptake. In contrast, lysozyme in combination with hypothiocyanous acid/hypothiocyanite without peroxidase gave only a 30-50% inhibition. The addition of 5 mmol/L dithiothreitol after incubation with lysozyme and hypothiocyanous acid/hypothiocyanite eliminated the inhibition of the bacterial glucose uptake. The viability of S. mutans was not affected by treatment with any of the components used. Our results indicate that physiological concentrations of lysozyme and the salivary peroxidase system components have a synergistic effect which results in a significant inhibition of glucose metabolism by S. mutans.

Characteristics of the Early Effect of Prolactin on Lactose Biosynthesis in Mouse Mammary Gland Explants

These studies were carried out to characterize the early effect of prolactin (PRL) on lactose biosynthesis in cultured mammary gland explants derived from 12- to 14-day pregnant mice. The rate of lactose biosynthesis was assessed by the rate of radiolabeled glucose incorporation into lactose. For the rapid isolation of lactose, a new method which involves the use of thin-layer chromatography on cellulose-impregnated plastic sheets was employed. The onset of the PRL stimulation of [3H]glucose incorporation into lactose occurred 6-8 hr after exposing the explants to PRL. The response to PRL was essentially all or none with maximum responses occurring with PRL concentrations above 25 ng/ml. The lowest stimulatory concentration of PRL was 10 ng/ml. The action of PRL on lactose biosynthesis requires both ongoing RNA and protein synthesis since puromycin, cyclohexamide, and actinomycin D abolished the PRL effect.

Studies of the distribution of glycogen between the inner cell mass and trophoblast cells of mouse embryos.

Autoradiographic and histochemical techniques were used to determine the localization of glycogen synthesized during in-vitro culture of preimplantation mouse embryos. During early cleavage embryos accumulated little glycogen and that which was synthesized was spread evenly in the blastomere cytoplasm. However, morula and early blastocyst stages accumulated relatively large amounts of glycogen, especially in the peripheral or trophoblastic cells in comparison to the inner cells or inner-cell-mass cells. Immunosurgical techniques were used to study the incorporation of radiolabelled glucose into the biochemical pools of inner-cell-mass and trophoblastic cells during culture for 24 h. In general, trophoblastic cells incorporated considerably more isotope than did inner-cell-mass cells, especially into the acid-soluble glycogen fraction. However, inner cell masses isolated on Day 4 of pregnancy incorporated more glucose into acid-soluble glycogen than did inner cells isolated from blastocysts at the end of culture for 24 h in isotope.