Transport,metabolism and distribution space of octanoate in the perfused rat liver

The scope of the present work was to investigate the metabolism and the passage of octanoate from albumin into the phospholipid bilayer of the plasma membrane and from thence into the cell space. The experiments were done in the isolated perfused rat liver with infusions of albumin and octanoate at various concentrations. Once steady-state conditions were attained, trace amounts of [1-¹⁴C]-octanoate, [¹³¹I]-albumin and [³H]-water were injected simultaneously and the effluent perfusate was fractionated. The normalized dilution curves were used for model analysis. The model which gives the best fit to the experimental results and which also produces the most consistent parameters is one that presupposes a rapid distribution of octanoate into the cell membrane and a slow transfer from the cell membrane into the cytosol. The concentration dependence of the distribution between the membrane and the extracellular space is parabolic, suggesting that octanoate changes the properties of the cell membrane when present at higher concentrations. The passage from the cell membrane into the cell space is relatively slow and limits metabolic transformation partly or totally, depending on the octanoate concentration in the plasma membrane. The rapid transfer of octanoate from the albumin space into the plasma membrane corroborates previous measurements of the dissociation of the albumin–octanoate complex. © 1997 John Wiley & Sons, Ltd.     

The actions of fisetin on glucose metabolism in the rat liver

Fisetin is a flavonoid dietary ingredient found in the smoke tree (Cotinus coggyria) and in several fruits and vegetables. The effects of fisetin on glucose metabolism in the isolated perfused rat liver and some glucose‐regulating enzymatic activities were investigated. Fisetin inhibited glucose, lactate, and pyruvate release from endogenous glycogen. Maximal inhibitions of glycogenolysis (49%) and glycolysis (59%) were obtained with the concentration of 200 µM. The glycogenolytic effects of glucagon and dinitrophenol were suppressed by fisetin 300 µM. No significant changes in the cellular contents of AMP, ADP, and ATP were found. Fisetin increased the cellular content of glucose 6‐phosphate and inhibited the glucose 6‐phosphatase activity. Gluconeogenesis from lactate and pyruvate or fructose was inhibited by fisetin 300 µM. Pyruvate carboxylation in isolated intact mitochondria was inhibited (IC₅₀ = 163.10 ± 12.28 µM); no such effect was observed in freeze‐thawing disrupted mitochondria. It was concluded that fisetin inhibits glucose release from the livers in both fed and fasted conditions. The inhibition of pyruvate transport into the mitochondria and the reduction of the cytosolic NADH‐NAD⁺ potential redox could be the causes of the gluconeogenesis inhibition. Fisetin could also prevent hyperglycemia by decreasing glycogen breakdown or blocking the glycogenolytic action of hormones. Copyright © 2010 John Wiley & Sons, Ltd.     

Effects of ranolazine on fatty acid transformation in the isolated perfused rat liver

It has been proposed that in the heart, ranolazine shifts the energy source from fatty acids to glucose oxidation by inhibiting fatty acid oxidation. Up to now no mechanism for this inhibition has been proposed. The purpose of this study was to investigate if ranolazine also affects hepatic fatty acid oxidation, with especial emphasis on cell membrane permeation based on the observations that the compound interacts with biological membranes. The isolated perfused rat liver was used, and [1-¹⁴C]oleate transport was measured by means of the multiple-indicator dilution technique. Ranolazine inhibited net uptake of [1-¹⁴C]-oleate by impairing transport of this fatty acid. The compound also diminished the extra oxygen consumption and ketogenesis driven by oleate and the mitochondrial NADH/NAD⁺ ratio, but stimulated ¹⁴CO₂ production. These effects were already significant at 20 μM ranolazine. Ranolazine also inhibited both oxygen consumption and ketogenesis driven by endogenous fatty acids in substrate-free perfused livers. In isolated mitochondria ranolazine inhibited acyl-CoA oxidation and β-hydroxybutyrate or α-ketoglutarate oxidation coupled to ADP phosphorylation. It was concluded that ranolazine inhibits fatty acid uptake and oxidation in the liver by at least two mechanisms: inhibition of cell membrane permeation and by an inhibition of the mitochondrial electron transfer via pyridine nucleotides.     

Ketogenesis evaluation in perfused liver of diabetic rats submitted to short‐term insulin‐induced hypoglycemia

Ketogenesis, inferred by the production of acetoacetate plus ß‐hydroxybutyrate, in isolated perfused livers from 24‐h fasted diabetic rats submitted to short‐term insulin‐induced hypoglycemia (IIH) was investigated. For this purpose, alloxan‐diabetic rats that received intraperitoneal regular insulin (IIH group) or saline (COG group) injection were compared. An additional group of diabetic rats which received oral glucose (gavage) (100 mg kg^?1) 15 min after insulin administration (IIH + glucose group) was included. The studies were performed 30 min after insulin (1.0 U kg^?1) or saline injection. The ketogenesis before octanoate infusion was diminished (p < 0.05) in livers from rats which received insulin (COG vs. IIH group) or insulin plus glucose (COG vs. IIH + glucose group). However, the liver ketogenic capacity during the infusion of octanoate (0.3 mM) was maintained (COG vs. IIH group and COG vs. IIH + glucose group). In addition, the blood concentration of ketone bodies was not influenced by the administration of insulin or insulin plus glucose. Taken together, the results showed that inspite the fact that insulin and glucose inhibits ketogenesis, livers from diabetic rats submitted to short‐term IIH which received insulin or insulin plus glucose showed maintained capacity to produce acetoacetate and ß‐hydroxybutyrate from octanoate. Copyright © 2009 John Wiley & Sons, Ltd.     

Compartmentation of [14C]Glutamate and [14C]Glutamine Oxidative Metabolism in the Rat Hippocampus as Determined by Microdialysis

Abstract: Metabolic compartmentation of amino acid metabolism in brain is exemplified by the differential synthesis of glutamate and glutamine from the identical precursor and by the localization of the enzyme glutamine synthetase in glial cells. In the current study, we determined if the oxidative metabolism of glutamate and glutamine was also compartmentalized. The relative oxidation rates of glutamate and glutamine in the hippocampus of free-moving rats was determined by using microdialysis both to infuse the radioactive substrate and to collect ¹⁴CO₂ generated during their oxidation. At the end of the oxidation experiment, the radioactive substrate was replaced by artificial CSF, 2 min-fractions were collected, and the specific activities of glutamate and glutamine were determined. Extrapolation of the specific activity back to the time that artificial CSF replaced ¹⁴C-amino acids in the microdialysis probe yielded an approximation of the interstitial specific activity during the oxidation. The extrapolated interstitial specific activities for [¹⁴C]glutamate and [¹⁴C]glutamine were 59 ± 18 and 2.1 ± 0.5 dpm/pmol, respectively. The initial infused specific activities for [U-¹⁴C]glutamate and [U-¹⁴C]glutamine were 408 ± 8 and 387 ± 1 dpm/pmol, respectively. The dilution of glutamine was greater than that of glutamate, consistent with the difference in concentrations of these amino acids in the interstitial space. Based on the extrapolated interstitial specific activities, the rate of glutamine oxidation exceeds that of glutamate oxidation by a factor of 5.3. These data indicate compartmentation of either uptake and/or oxidative metabolism of these two amino acids. The presence of [¹⁴C]glutamine in the interstitial space when [¹⁴C]glutamate was perfused into the brain provided further evidence for the glutamate/glutamine cycle in brain.     

Metabolic effects of acetate in perfused rat liver studies on ketogenesis,glucose output,lactate uptake and lipogenesis

1. Livers from fed male rats were perfused in situ in a non-recirculating system with whole rat blood containing acetate at six concentrations, from 0.04 to 1.5 μmol/ml, to cover the physiological range encountered in the hapatic portal venous blood in vivo. 2. Below a concentration of 0.25 μmol/ml there was net production of acetate by the liver, while above it there was ner uptake with a fractional extraction of 40%. 3.No relationship was observed between blood [acetate] and hepatic ketogenesis, the ration [3-hydroxybutyrate]/[acetoacetate] or glucose output, either at low fatty acid concentration s or during oleate infusion. 4. Following the increase in serum fatty acid concentration, induced by oleate infusion, there were suquential incresase in ketogenesis and the ratio of [3-hydroxybutyrate]/[acetoacetate] while glucose output rose and lactate uptake fell significantly after in redox state. 5. There was a highly significant negative correlation between blood [acetate] and hepatic lactate uptake during oleate infusion. At the highest acetate concentration of 1.5 μmol/ml there was a small net hepatic lactate output. After oleate infusion ceased, lactate uptake increased, but the negative correlation between blood [acetate] and hepatic lactate uptake persisted. 6. Livers were also perfused with iether [1-¹⁴C]acetate or [U-¹⁴C]lactate at a concentration of acetate of either 0.3 or 1.3 μmol/ml of blood. With [1-¹⁴C]acetate, most of the radioactivity was recovered as fatty acids at the lower concentration of blood acetate. At the higher blood [acetate] a considerably smaller proportion of the radioactivity was recovered in lipids. With [U-¹⁴C]lactate the reverse pattern obtained i.e., recovery was greater at the high concentration of acetate and fell at the low concentration. Fatty acid biosynthesis, measured with ³H₂O, was stimulated from 2.4 to 6.6 μmol of fatty acid/g of liver per h by high blood [acetate] although the contribution of (acetate+lactate) to synthesis remained constant at 33–38% of the total. 7. These results emphasize the important role of the liver in regulating blood acetate concentrations and indicate that it can be major hepatic substrate. Acetate taken up by the liver appeared to compete directly with lactate, for lipogenesis and metabolism and acetate uptake was inhibited by raised bloodd [lactate].     

Lacto-N-neotetraosylceramide ("paragloboside") as a possible tumor-associated surface antigen of hamster NILPY tumor.

Isolated rat hepatocytes from fed and starved rats synthesized net glucose from various precursors at similar rates. [3-¹⁴C]-lactate incorporation into glucose was also similar in hepatocytes from fed and starved rats, as was ketone body formation from oleate and octanoate. Rates of gluconeogenesis in hepatocytes from fed rats compare to rates seen in perfused livers from starved rats rather than perfused livers from fed rats. Thus metabolic rates and possibly controls may be different between perfused livers and isolated hepatocytes when using fed rats.     

Metabolism of [3H]leupeptin by rat liver

Tritium-labeled leupeptin was used to study how this tripeptide proteinase inhibitor interacts with the liver, including the mechanism of its transport into the cell, its subcellular distribution after uptake, and its metabolism once in the tissue. Experiments were done in situ and in a perfused liver. At low concentrations (1 to 10 μm) the uptake of radioactive inhibitor was competed by chemically reduced leupeptin. At high concentrations at least up to 400 μm the uptake was directly proportional to the external concentration of tripeptide. Entry into the tissue essentially stopped at low temperature (<21 °C). [³H]Leupeptin initially was located in the soluble fraction of the liver homogenate and by 15 to 30 min became concentrated in the lysosome-rich fraction. During 2 h of perfusion almost 50% of [³H]leupeptin that had entered the liver was secreted intact into the bile. In addition, a portion of the leupeptin that remained in the liver was degraded during this time period.     

Higher Free Fatty Acid Uptake in Visceral Than in Abdominal Subcutaneous Fat Tissue in Men

Visceral adipose tissue has been shown to have high lipolytic activity. The aim of this study was to examine whether free fatty acid (FFA) uptake into visceral adipose tissue is enhanced compared to abdominal subcutaneous tissue in vivo. Abdominal adipose tissue FFA uptake was measured using positron emission tomography (PET) and [¹⁸F]‐labeled 6‐thia‐hepta‐decanoic acid ([¹⁸F]FTHA) and fat masses using magnetic resonance imaging (MRI) in 18 healthy young adult males. We found that FFA uptake was 30% higher in visceral compared to subcutaneous adipose tissue (0.0025 ± 0.0018 vs. 0.0020 ± 0.0016 µmol/g/min, P = 0.005). Visceral and subcutaneous adipose tissue FFA uptakes were strongly associated with each other (P < 0.001). When tissue FFA uptake per gram of fat was multiplied by the total tissue mass, total FFA uptake was almost 1.5 times higher in abdominal subcutaneous than in visceral adipose tissue. In conclusion, we observed enhanced FFA uptake in visceral compared to abdominal subcutaneous adipose tissue and, simultaneously, these metabolic rates were strongly associated with each other. The higher total tissue FFA uptake in subcutaneous than in visceral adipose tissue indicates that although visceral fat is active in extracting FFA, its overall contribution to systemic metabolism is limited in healthy lean males. Our results indicate that subcutaneous, rather than visceral fat storage plays a more direct role in systemic FFA availability. The recognized relationship between abdominal visceral fat mass and metabolic complications may be explained by direct effects of visceral fat on the liver.     

Determination of nucleic acid by [tetra‐(3‐methoxy‐4‐hydroxyphenyl)]–Tb3+ porphyrin as the fluorescence spectral probe in bis(2‐ethylhexyl)sulfosuccinate sodium salt micelle system

A new system for the determination of nucleic acid by rare earth metallic porphyrin of [tetra‐(3‐methoxy‐4‐hydroxyphenyl)]–Tb³⁺ [T(3‐MO‐4HP)–Tb³⁺] porphyrin as fluorescence spectral probe has been developed in this paper. Nucleic acid can enhance the fluorescence intensity of the T(3‐MO‐4HP)–Tb³⁺ porphyrin in the presence of bis(2‐ethylhexyl)sulfosuccinate sodium salt(AOT) micelle. In pH 8.00 Tris–HCl buffer solution, under optimum conditions, the enhanced fluorescence intensity is in proportion to the concentration of nucleic acids in the range of 0.05–3.00 µg mL^?1 for calf thymus DNA (ct DNA) and 0.03–4.80 µg mL^?1 for fish sperm DNA(fs DNA). Their detection limits are 0.03 and 0.01 µg mL^?1, respectively. In addition, the binding interaction mechanism between T(3‐MO‐4HP)–Tb³⁺ porphyrin and ct DNA is also investigated by resonance scattering and fluorescence spectra. The maximum binding number is calculated by molar ratio method. The new system can be used for the determination of nucleic acid in pig liver, yielding satisfactory results. Copyright © 2009 John Wiley & Sons, Ltd.     

Origins of the Extracellular Glutamate Released During Total Metabolic Blockade in the Immature Retina

Abstract: Previous studies have shown that complete blockade of metabolism in embryonic chick retina causes a time-dependent increase in the release of glutamate into the extracellular space. The present study examined the cellular source of this glutamate, i.e., neuronal and/or glial. Pure cultures of retinal neurons or glia were labeled for 10 min at 37°C with [³H]acetate. Retinal glia, but not retinal neurons, were found to selectively and preferentially metabolize acetate, thus producing ³H-labeled amino acids in the glial compartment. This finding provides direct evidence to substantiate findings from several other laboratories that have indirectly determined the preferential metabolism of acetate by glia by using mixed neuronal/glial populations. To study the cellular source of glutamate released during total metabolic blockade, whole retina were prelabeled with [³H]acetate plus [U-¹⁴C]glucose (to label the neuronal compartment). Total metabolic blockade was instituted with a combination of iodoacetate (IOA) plus KCN, and the release of glutamate into the medium was followed at 5, 15, and 30 min. During total energy blockade, net extracellular glutamate was not elevated at 5 min [0.17 ± 0.02 vs. 0.12 ± 0.01 µM for treated vs. control retina (means ± SEM), respectively], but was increased significantly at 15 (1.2 ± 0.26 µM) and 30 min (2.6 ± 0.22 µM). Total [³H]glutamate in the medium during IOA/KCN treatment was unchanged at 5 min, but was increased 1.5- and threefold above basal levels at 15 and 30 min, respectively. During the time when extracellular glutamate increased, the specific activity of [³H]glutamate remained fairly constant, 731 ± 134 and 517 ± 82 dpm/nmol (means ± SEM) at 15 and 30 min, respectively. In contrast, ¹⁴C-labeled glutamate in the medium did not increase during IOA/KCN treatment and paralleled basal levels. Thus, the specific activity of ¹⁴C-labeled extracellular glutamate decreased from 309 ± 87 dpm/nmol at 15 min to 42 ± 8 dpm/nmol at 30 min. Prior loading of the tissue with 0.5 mM trans-pyrrolidine-2,4-dicarboxylate (t-PDC), a glutamate transport inhibitor, blocked 57% of the glutamate released at 30 min of IOA/KCN exposure, suggesting that reversal of an Na⁺-dependent glutamate transporter was a key contributor to the appearance of extracellular glutamate during energy deprivation. The increase in extracellular [³H]glutamate, constancy of the specific activity of extracellular [³H]glutamate, decrease in the specific activity of extracellular [¹⁴C]glutamate, and attenuation of release by prior loading with t-PDC indicate that glial pools of glutamate released via reversal of the transporter contribute significantly to the rise in extracellular glutamate after metabolic inhibition in this preparation.     

Oxidation of branched-chain amino acids in skeletal muscle and liver of rat Effects of octanoate and energy state

The effect of octanoate on the oxidative decarboxylation of ¹⁴C-labeled amino acids has been studied in perfused hindquarter and liver of rat. Regulation of the branched-chain α-keto acid dehydrogenase has been further studied with α-[¹⁴C-1]ketoisovalerate in isolated rat muscle and liver mitochondria. (1) Octanoate has a stimulatory effect on the oxidation of branched-chain amino acids in perfused hindquarter. The oxidative decarboxylation of other amino acids are inhibited. Octanoate inhibits the oxidative decarboxylation of all amino acids in perfused liver. (2) The oxidation of valine is stimulated by octanoate and hexanoate also in isolated muscle mitochondria. The stimulatory effect is probably related to activation of the fatty acids since acyl-carnitines inhibit the oxidation. (3) The oxidation of α-ketoisovalerate in mitochondria is inhibited by competing substrates (pyruvate, α-ketoglutarate and succinate). This inhibition is counteracted by octanoate and ADP. (4) Low concentrations (1–5 μM) of 2,4-dinitrophenol (DNP) activates wheras higher concentrations inactivates the branched-chain α-keto acid dehydrogenase in intact but not in solubilized muscle mitochondria. The inactivation is counteracted by ATP, but is increased by octanoate. (5) The observations seem to suggest that the activation (like the inactivation) of branched-chain α-keto acid dehydrogenase in skeletal muscle is dependent on the mitochondrial energy state which therefore may regulate both activation and inactivation of the dehydrogenase.     

Glucose transport and metabolism in the perfused hindquarters of lean and obese-hyperglecemic (db/db) mice effects of insulin and electrical stimulation

Changes in glucose transport and metabolism in skeletal muscles of the obese-diabetic mice (db/db) was characterized using the perfused mouse hindquarter preparation. Metabolism of [5-³H]glucose, uptake of 3-O-[methyl-³H]glucose (methylglucose) and [2-¹⁴C]deoxyglucose (deoxyglucose) was studied under resting, electrically stimulated contracting, and insulin-stimulated conditions. Basal rate of methylglucose uptake was 255 ± 18 and 180 ± 9 μl/15 min per ml intracellular fluid space for lean and db/db mice, respectively. The V_? of methylglucose transport was decreased with no change in K_m in the db/db mice. Both electrical stimulation and insulin (1/mU/ml) increased methylglucose uptake rate 2-fold in both lean and obese mice. We observed no significant change in insulin sensitivity in the db/db mice in stimulating methylglucose uptake which was subnormal under all conditions. Similar results were obtained using deoxyglucose. Likewise, uptake of glucose and ³H₂O production from [5-³H]glucose were significantly reduced, both at rest and during electrically stimulated contraction in the db/db mouse. However, lactate production in the electrically stimulated db/db mouse preparations was not significantly different from that in the lean mice. These data suggest a major contribution from an impaired glucose transport activity to the reduction in glucose metabolism in the db/db mouse skeletal muscle.     

Uptake and metabolism of d‐glucose in isolated acinar and ductal cells from rat submandibular glands

The present study deals with the possible effects of selected environmental agents upon the uptake and metabolism of d ‐glucose in isolated acinar and ductal cells from the rat submandibular salivary gland. In acinar cells, the uptake of d ‐[U‐¹⁴C]glucose and its non‐metabolised analogue 3‐O‐[¹⁴C‐methyl]‐d ‐glucose was not affected significantly by phloridzin (0.1 mM) or substitution of extracellular NaCl (115 mM) by an equimolar amount of CsCl, whilst cytochalasin B (20 μM) decreased significantly such an uptake. In ductal cells, both phloridzin and cytochalasin B decreased the uptake of d ‐glucose and 3‐O‐methyl‐d ‐glucose. Although the intracellular space was comparable in acinar and ductal cells, the catabolism of d ‐glucose (2.8 or 8.3 mM) was two to four times higher in ductal cells than in acinar cells. Phloridzin (0.1 mM), ouabain (1.0 mM) and cytochalasin B (20 μM) all impaired d ‐glucose catabolism in ductal cells. Such was also the case in ductal cells incubated in the absence of extracellular Ca²⁺ or in media in which NaCl was substituted by CsCl. It is proposed that the ductal cells in the rat submandibular gland are equipped with several systems mediating the insulin‐sensitive, cytochalasin B‐sensitive and phloridzin‐sensitive transport of d ‐glucose across the plasma membrane. Copyright © 2014 John Wiley & Sons, Ltd.     

Unidirectional uptake of enkephalins at the blood-tissue interface of the blood-cerebrospinal fluid barrier: a saturable mechanism

The cellular uptake at the blood-tissue interface of the blood-cerebrospinal fluid (CSF) barrier to tyrosyl-3,5-[³H]enkephalin-[5-l-leucine] (abbreviated to Leu-enkephalin) and of its synthetic analogue d-alanine²-tyrosyl-3,5-[³H]enkephalin-[5-d-leucine] (abbreviated to d-Ala²-d-Leu⁵-enkephalin) was studied in the isolated perfused choroid plexuses from the lateral ventricles of the sheep, using the rapid (<30 s), single circulation, paired-tracer dilution technique, in which d-[¹⁴C]-mannitol serves as an extracellular marker. Cellular uptake of peptides was estimated by directly comparing venous dilution profiles of [³H] and [¹⁴C] radioactivities in the absence and presence of unlabelled peptide, the N-terminal amino acid (l-tyrosine), the typical l-transport system substrate, 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) and the inhibitor of aminopeptidase activity, bacitracin. The cellular uptake of both enkephalins was strongly (65–76%) but not completely inhibited by the addition of 5 mM unlabelled peptide to the bolus; the self-inhibition was significantly higher for d-Ala²-d-Leu⁵-enkephalin than for Leu-enkephalin. The addition to the bolus of l-tyrosine (5 mM), BCH (10 mM) or bacitracin (2 mM) reduced the ³H-radioactivity uptake by the choroid plexus of both enkephalins by 20–40%, the degree of inhibition being greater for [³H]-Leu-enkephalin than for its analogue.It is concluded that during single passage of enkephalins through the choroid plexus circulation, unidirectional uptake at the blood-tissue interface of the blood-CSF barrier consists of two components; a saturable component, which represents uptake of the intact peptide by the choroid epithelium, and a non-saturable component, which reflects enzymatic degradation of peptide in the blood and/or at the barrier, with a liberation of the N-terminal tyrosyl residue. Higher penetration of the blood-CSF barrier by d-Ala²-d-Leu⁵-enkephalin can be attributed to its greater resistance to hydrolysis.     

Flow rates in the isolated perfused rat liver by pulse labeling with radioactive substrates and mathematical analysis of the wash-out kinetics

The multiple indicator dilution technique (Goresky and Bach) is critically evaluated and its application to the isolated, hemoglobin-free perfused rat liver is described. From the results of pulse labelling experiments using indicator substances for the total aqueous space of the liver ([3H]water and [14C]urea) an for the extracellular space ([14C]sucrose, [3H]-inulin and [3H]dextrane), it is concluded that the mathematical model of the liver in situ according to Goresky and Bach is sufficient to also describe the hemodynamics of the isolated liver perfused with a saline solution. The data indicate that the multiple indicator dilution technique in combination with the available mathematical basis is applicable to the study of transport across the liver cell membrane. The method, however, is restricted to compounds which are metabolized very slowly, such as D-lactate. A possible extension of this method to metabolic processes is discussed in view of pulse labelling experiments with [3H]-and [1-14C]L-lactate. Since the involvement of metabolism in the available mathematical model is not differentiated and the release of products is not taken into consideration, the method in its present state is not applicable to studies of metabolism. Moreover, even the parameters of transport derived by this technique are of limited value, when the transported compounds are rapidly metabolized by reversible reactions. Despite these uncertainties, the present data indicate that the transport of L-lactate is ten times faster than that of D-lactate.     

Plant-originated glycoprotein (24 kDa) has an inhibitory effect on proliferation of BNL CL.2 cells in response to di(2-ethylhexyl)phthalate

Di(2‐ethylhexyl)phthalate (DEHP) is one of the many environmental chemicals that are widely used in polyvinyl chloride products, vinyl flooring, food packaging and infant toys. They cause cell proliferation or dysfunction of human liver. The purpose of this study is to investigate the inhibitory effect of a glycoprotein (24 kDa) isolated from Zanthoxylum piperitum DC (ZPDC) on proliferation of liver cell in the DEHP‐induced BNL CL. 2 cells. [³H]‐thymidine incorporation, intracellular reactive oxygen species (ROS), intracellular Ca²⁺ mobilization and activity of protein kinase C (PKC) were measured using radioactivity and fluorescence method respectively. The expression of mitogen‐activated protein kinases [extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK)], activator protein (AP)‐1 (c‐Jun and c‐Fos), proliferating cell nuclear antigen (PCNA) and cell cycle‐related factors (cyclin D1/cyclin‐dependent kinase [CDK] 4) were evaluated using Western blotting or electrophoretic mobility shift assay. The results in this study showed that the levels of [³H]‐thymidine incorporation, intracellular ROS, intracellular Ca²⁺ mobilization and activity of PKCα were inhibited by ZPDC glycoprotein (100 µg/ml) in the DEHP‐induced BNL CL. 2 cells. Also, activities of ERK, JNK and AP‐1 were reduced by ZPDC glycoprotein (100 µg/ml). With regard to cell proliferation, activities of PCNA and cyclin D1/CDK4 were significantly suppressed at treatment with ZPDC glycoprotein (100 µg/ml) in the presence of DEHP. Taken together, these findings suggest that ZPDC glycoprotein significantly normalized activities of PCNA and cyclin D1/CDK4, which relate to cell proliferation factors. Thus, ZPDC glycoprotein appears to be one of the compounds derived from natural products that are able to inhibit cell proliferation in the phthalate‐induced BNL CL. 2 cells. Copyright © 2011 John Wiley & Sons, Ltd.     

Extraction of estrogens by the hind limb of the dog. Evidence for entry into the lymphatics.

Substantial extrasplanchnic metabolism of estrogens is known to occur in humans and dogs. As part of an investigation into the anatomic sites of such metabolism, the extraction of estrogens by the hind limb of the dog was studied during a constant infusion of [³H]estrone. Simultaneous femoral artery (A) and femoral vein (FV) plasma samples were obtained and analyzed for total radioactivity, unconjugated and conjugated radioactivity, for [³H]estrone and for its metabolites estradiol-17β, estrone sulfate and estrone glucosiduronate. The percent extraction across the hind limb was calculated [100(1-FV/A)]. The mean percent extraction ± SE of total, conjugated and unconjugated radioactivity was 31 ± 3.9, 27 ± 4.4 and 16 ± 3.7 respectively, indicating significant net uptake of these moieties by the hind limb (P<.01). Mean percent extractions ± SE for estrone and estradiol-17β were 40 ± 4.9 and 32 ± 2.7, indicating significant net uptake of these specific unconjugated estrogens by the hind limb (P<.01). The mean percent extraction of estrone glucosiduronate was 16 ± 3.1 indicating significant net uptake of this conjugate (P<.01). However, the mean percent extraction of estrone sulfate was negative (?12 ± 4.1) indicating net production of this conjugate by the hind limb (P<.01). Since the net uptake of total radioactivity cannot be explained on the basis of metabolism by the hind limb, the lymphatics were investigated as an alternate efferent pathway. In similar experiments the thoracic duct was cannulated, the estrogens in lymph were analyzed and compared with those in femoral artery plasma. Each estrogen measured in plasma appeared in lymph within 10 minutes following the start of the [³H]estrone infusion. The lymph/femoral artery concentration ratios reached a plateau at 70–100 minutes after the start of the infusion. The plateau concentrations were 20–70% of those in plasma. It is suggested that removal of estrogens in the lymph may account, in part at least, for the net uptake of total radioactivity across the hind limb calculated from the plasma data.     

Productivity of Chlorella sorokiniana in a short light‐path (SLP) panel photobioreactor under high irradiance

Maximal productivity of a 14 mm light‐path panel photobioreactor under high irradiance was determined. Under continuous illumination of 2,100 µmol photons m^?2 s^?1 with red light emitting diodes (LEDs) the effect of dilution rate on photobioreactor productivity was studied. The light intensity used in this work is similar to the maximal irradiance on a horizontal surface at latitudes lower than 37°. Chlorella sorokiniana, a fast‐growing green microalga, was used as a reference strain in this study. The dilution rate was varied from 0.06 to 0.26 h^?1. The maximal productivity was reached at a dilution rate of 0.24 h^?1, with a value of 7.7 g dw m^?2 h^?1 (m² of illuminated photobioreactor surface) and a volumetric productivity of 0.5 g dw L^?1 h^?1. At this dilution rate the biomass concentration inside the reactor was 2.1 g L^?1 and the photosynthetic efficiency was 1.0 g dw mol photons. This biomass yield on light energy is high but still lower than the theoretical maximal yield of 1.8 g mol photons^?1 which must be related to photosaturation and thermal dissipation of absorbed light energy. Biotechnol. Bioeng. 2009; 104: 352–359 © 2009 Wiley Periodicals, Inc.     

THE INFLUENCE OF PORTOCAVAL ANASTOMOSIS ON THE METABOLISM OF LABELLED OCTANOATE, BUTYRATE AND LEUCINE IN RAT BRAIN

Rats were given a portocaval anastomosis and 3 weeks later, when the only ultrastructural change in the CNS is watery swelling of astrocytes, several aspects of brain metabolism were studied. The uptake of leucine by the brain, its incorporation into protein and its oxidation were followed after the simultaneous injection of a mixture of L-[1¹⁴C]leucine and L-[4,5-³H]leucine. The concentration of leucine in blood was lowered in the operated animals whereas in brain it was increased. The specific radioactivity of leucine in the brain was comparable to values in control animals and there was no evidence of a decrease in incorporation of [1-¹⁴C]leucine into brain proteins over the short experimental time period studied. The only difference from the controls in the oxidation of [4,5-³H]leucine was a greater accumulation in glutamine. The amount of glutamine in the brains of the operated animals had increased 4-fold at the time of the metabolic studies. From dual-labelled experiments in which a mixture containing [1-¹⁴C]butyrate and L-[4,5-³H]leucine was injected intravenously, it was shown that, in both control and operated animals, the pools of brain glutamate and glutamine labelled from butyrate were metabolically distinct from those labelled from leucine. The total radioactivity appearing in brain from [1-¹⁴C]butyrate was markedly reduced in operated animals, but the radioactivity from L-[4,5-³H]leucine was not. The metabolism of [1-¹⁴C]octanoate was compared with that of [1-¹⁴C]butyrate. In control animals the labelling of metabolites was almost identical with either precursor. In operated animals there was no reduction in the uptake of [1-¹⁴C]octanoate into the brain. There was evidence that the size of the glutamine pool labelled, relative to glutamate, was increased but that it had a slower fractional turnover coefficient. A link between astroglial changes and an impairment to the carrier mechanism for transport of short chain monocarboxylic acids across the blood-brain barrier is suggested.