Role of glutamate dehydrogenase reaction in the control of citrate pool in yeast

• 1.1. Role of NADP-glutamate dehydrogenase in the depletion of citrate was analyzed using permeabilized yeast cells.
• 2.2. Citrate was converted to 2-oxoglutarate, which was then metabolized to glutamate by NADP-glutamate dehydrogenase in the presence of ammonium ion.
• 3.3. Formation of 2-oxoglutarate plus glutamate was in good agreement with the concentration of citrate decreased. Glutamate formation can be a good indicator of the depletion of citrate, because 70% of the citrate decreased was converted to glutamate.
• 4.4. Glycolytic activity was closely correlated with the decrease in citrate under the in situ conditions.
• 5.5. NADP-glutamate dehydrogenase increased in anaerobically grown yeast cells.
• 6.6. An effective depletion of citrate by increased synthesis of NADP-glutamate dehydrogenase can explain the lowered mechanism of citrate causing glycolytic stimulation under the anaerobic growth conditions of yeast.

     

Effect of methyl methacrylate on mitochondrial function and structure

• 1.1. Treatment of isolated rat liver mitochondria with methyl methacrylate (MM) produced membrane disruption as evidenced by the release of citrate synthase, and changes in the ultrastructure of mitochondria.
• 2.2. At concentration 0.1%, MM uncoupled oxidative phosphorylation as evidenced by stimulation of state 4 respiration supported either by pyruvate plus malate or succinate (+rotenone) and ATP-ase activity in intact mitochondria.
• 3.3. At concentration 1% MM stimulated ATP-ase activity in intact mitochondria and succinate (+rotenone) oxidation at state 4 and was without effect on this substrate oxidation at state 3.
• 4.4. MM inhibited pyruvate plus malate oxidation either at state 3 or in the presence of uncoupling agents.
• 5.5. MM inhibited the NADH oxidase of electron transport particles at a concentration which failed to inhibit either succinic oxidase or the NADH-ferricyanide reductase activity.
• 6.6. The data presented suggest that in the isolated mitochondria MM inhibits NADH oxidation in the vicinity of the rotenone sensitive site of complex I.
• 7.7. The general conclusion is that MM may block an electron transport and to uncouple oxidative phosphorylation in rat liver mitochondria. The overall in vitro effect would be to prevent ATP synthesis which could result in cell death under in vivo conditions.

     

Alteration of 5-hydroxytryptophan-induced hypothermia by serotonergic agents in the adult domestic fowl (Gallus domesticus)

• 1.1.|5-Hydroxytryptophan (5-HTP) induced a dose-dependent hypothermia in adult fowls.
• 2.2.|The hypothermic effect of 5-HTP was potentiated by carbidopa, citalopram, additive with (±), (−) and (+) propanolol and antagonised by methysergide and metitepine.
• 3.3.|Cyproheptadine, xylamidine and ketanserin did not antagonised 5-HTP-induced hypothermia.
• 4.4.|The results suggest that the hypothermic effect of 5-HTP in fowls may be mediated mainly via activation of central 5-HT receptors, probably 5-HT₁ receptors.

     

The effect of d2o on glycolysis by rat hepatocytes

• 1.1. The effect of incorporating D₂O into the incubation medium on glycolysis and gluconeogenesis by hepatocytes from fasted rats was examined.
• 2.2. The substitution by heavy water, D₂O, at concentrations from 10 to 40%, stimulated glucose uptake, lactate production and CO₂ yields from glucose. At 10 mM glucose, 40% D₂O doubled glucose uptake, increased CO₂ production by 40%, and increased lactate production by 350%.
• 3.3. The stimulation of lactate production decreased at higher glucose concentrations, but was still substantial even at 80 mM glucose.
• 4.4. There was no effect on CO₂ production above glucose concentrations of 30 mM.
• 5.5. Ten percent D₂O showed little inhibition of lactate uptake, its oxidation and gluconeogenesis. At 40% D₂O the inhibition ranged from 10 to 20%.
• 6.6. No effect of D₂O on the rate of glucokinase or glucose-6-phosphatase was observed.
• 7.7. The concentration of fructose, 2,6-P was not affected by D₂O

     

A simple and reliable method for the purification of Pseudomonas aeruginosa phospholipase C produced in a high phosphate medium containing choline

• 1.1. Pseudomonas aeruginosa phospholipase C from culture supernatants of bacteria grown in high-P_i basal salt medium with choline, as the sole carbon and nitrogen source, was purified by precipitation with 70% saturation ammonium sulfate in the presence of celite.
• 2.2. The PLC activity was eluted of this mixture by the use of a reverse gradient of 70-0% ammonium sulfate.
• 3.3. The peak containing the PLC activity revealed a single protein after SDS-PAGE.
• 4.4. The method could also be applied to purify PLC produced in a low-P_i complex medium. The resultant preparation was not homogeneous.
• 5.5. The molecular weight for both PLC preparations was about 70 kDa.
• 6.6. Both PLC used phosphatydilcholine and sphingomyelin as substrates, displayed hemolytic activity an exhibited an apparent K_M of 25 mM for p-nitrophenylphosphorylcholine.
• 7.7. They were not inhibited by 1% sodium deoxycholate but were 30% inhibited by 1% Triton X-100.
• 8.8. 2% sodium dodecylsulfate and 1% tetradecyltrimethylammonium bromide inhibited the PLC from the HP_l-BSM plus choline but not the enzyme from the LP_l-CM.

     

Fetal lung metabolism: Response to maternal fasting

• 1.1. Fetal lung metabolic response to maternal fasting late in gestation was investigated.
• 2.2. Maternal fasting 4 days before term was associated with low fetal plasma glucose and insulin levels but increased levels of fetal plasma glucagon, glycerol, lactate and fatty acids.
• 3.3. Fetuses from fasted mothers showed a significant decrease in body weight (30%), lung weight (30%) and lung glycogen (46%), but no change in lung protein, phospholipid or total lung DNA, suggesting that lung size is affected more than maturation.
• 4.4. Fetal lung slices incubated in vitro showed that lactate oxidation to CO₂ equalled that of glucose in control fetal lungs and was unaffected by maternal fasting, while glucose oxidation was depressed (23%).
• 5.5. Maternal fasting significantly decreased in vitro incorporation of [U-¹⁴C]-glucose, [U-¹⁴C]lactate and [1-¹⁴C]palmitate into lung phospholipids.
• 6.6. Fetal lungs from fasted mothers showed increased conversion of lactate to glucose, indicating gluconeogenic potential by fetal lung.
• 7.7. These studies show that plasma lactate serves as an important energy fuel and substrate for lipid synthesis for the fetal lung, and maternal fasting markedly alters fetal lung metabolism.

     

Metabolic energy and cytoskeletal requirements for synthesis and secretion by acini from rat mammary gland—ii. Intracellular transport and secretion of protein and lactose

• 1.1. Iodoacetate, 2,4-dinitrophenol, cyanide and cycloheximide inhibited protein secretion as well as synthesis by acini (alveoli) from rat mammary gland. Cytochalasin B and vinblastine inhibited protein secretion and marginally reduced protein synthesis. Colchicine was without effect on protein synthesis but inhibited secretion.
• 2.2. Intracellular protein transport was altered during incubation with metabolic and cytoskeletal inhibitors. Cycloheximide, iodoacetate. 2,4-dinitrophenol and Cytochalasin B appeared to block protein synthesis on polysomes of rough endoplasmic reticulum. Vinblastine inhibited protein transport from rough endoplasmic reticulum to Golgi apparatus and colchicine appeared to cause accumulation of protein in several endomembrane fractions.
• 3.3. Iodoacetate reduced acinar lactose content but was without effect on lactose synthetase activity. Cyanide, cycloheximide and vinblastine reduced lactose synthetase activity but not tissue lactose concentration. Cytochalasin B reduced glucose incorporation but was without effect on lactose content and lactose synthetase activity. Colchicine and 2,4-dinitrophenol did not alter glucose incorporation, lactose content or lactose synthetase activity. Lactose secretion was inhibited by all metabolic and cytoskeletal inhibitors examined.
• 4.4. Results indicated that sustained protein secretion depended on continued protein synthesis and that lactose secretion was coupled to protein secretion.

     

The effects of oxalate and glucose on lipogenesis by isolated hepatocytes from normal and biotin-deficient chicks (Gallus domesticus)

• 1.1. Isolated hepatocytes synthesize fatty acids and cholesterol from lactate and acetate with lactate being the more effective substrate.
• 2.2. Biotin deficiency decreased fatty add synthesis from both substrates but stimulated cholesterogenesis.
• 3.3. Exposure of intact hepatocytes to oxalate inhibited fatty acid and cholesterol synthesis from lactate, this effect was enhanced in biotin-deficient chicks. A similar effect was not observed when acetate was the substrate.
• 4.4. Synthesis of fatty acids from lactate and acetate was stimulated by glucose, biotin deficiency increased this response. Cholesterogenesis was reduced in control but not biotin-deficient chicks.

     

Aerobic glucose disposal in the oyster: An in vivo kinetic analysis

• 1.1. Aerobic glucose disposal in starved oysters exposed to 1 mM external glucose was 2.29 μg C/g wet wt/min.
• 2.2. It was hypothesized that the maximum disposal rate is limited by the maximum rate of transepithelial glucose transport.
• 3.3. The major recipients of glucose-carbon were glycogen and amino acids. 4. The rate of glucose-carbon disposal to these two pools was 0.80 and 0.42 μg C/g/min, respectively.
• 4.5. The internal energy state determines the pathways of glucose disposal.
• 5.6. Disposal of glucose-carbon in “glucose-primed” oysters is primarily into glycogen.
• 6.7. In fasted bivalves the disposal is primarily into amino acids and carboxylic acids.
• 7.8. The uptake of dissolved glucose has the potential of contributing significantly to growth under conditions where the external glucose concentration is kept artificially high.

     

Interaction of lipogenic substrates in porcine adipose tissue in vitro

• 1.1. Porcine adipose tissue was incubated with radiolabeled glucose, acetate or lactate. Saturation curves indicated that lactate > glucose > acetate in providing two-carbon units for fatty-acid synthesis.
• 2.2. Competition between individual substrates indicated that lactate was the best lipogenic substrate.
• 3.3. Incubation of all three substrates at concentrations observable in serum indicated that at 5.56mM, glucose was the preferred lipogenic substrate in the presence of 0.1 mM acetate and 1.0 mM lactate.
• 4.4. At elevated concentrations (18.52mM glucose, 1.0 mM acetate and 10.0 mM lactate), acetate and lactate were preferred to glucose as lipogenic substrates.

     

Effect of adenosine on gluconeogenesis in the perfused liver of chicken and guinea-pig

• 1.1. Glucose formation from lactate by the perfused liver of 48 hr starved chickens was strongly inhibited by adenosine (Ado); the half-maximal inhibition was attained at 40 μM. This effect was paralleled by a four- to five-fold increase of ATP content as determined in freeze-clamped liver.
• 2.2. In chicken liver homogenate gluconeogenesis from precursors such as alanine, glutamate, glutamine and aspartate, which are not converted into glucose by the perfused chicken liver, proceeded at rates equal to or higher than that with lactate, being markedly inhibited by Ado.
• 3.3. In the perfused guinea-pig liver glucose synthesis with lactate, propionate, glycerol and fructose was also inhibited by Ado; however, when precursors such as pyruvate, glutamine and a mixture of lactate + pyruvate were supplied to the liver Ado did not inhibit gluconeogenesis.
• 4.4. Assay of adenine nucleotides in the perfused guinea-pig liver, stopped by freeze-clamping technique in a number of experimental variants, revealed no correlation between the rate of gluconeogenesis and the changes induced by Ado in the adenine nucleotide pool.
• 5.5. In the perfused liver of both chicken and guinea-pig Ado produced an increase of the lactate to pyruvate ratio and, in general, a diminution of the content of malate-aspartate shuttle intermediates.
• 6.6. The results are interpreted as suggesting that the inhibitory effect of Ado on hepatic gluconeogenesis is not necessarily mediated by the changes in the adenine nucleotide pool.

     

Some properties of ATPase activity in the intact cells of yeast Saccharomycopsis fibuligera

• 1.1. The properties of ATPase activity were studied with the cells at the early stationary phase of Saccharomycopsis fibuligera.
• 2.2. Optimal pH for the activity was approximately 7.
• 3.3. The activity was stimulated by Mg²⁺.
• 4.4. The activity was inhibited by NaF, DCCD, oligomycin, NaN₃, NaVO₃, or PCMB but not inhibited by ouabain.

     

Effects of colchicine on milk yield,composition, and cellular differentiation during caprine lactogenesis

• 1.1. Intramammary colchicine infusion into goats at parturition reduced milk yield by 20% during the 30 day experimental period.
• 2.2. During the first week of lactation, milk composition from colchicine-treated udder halves had elevated somatic cell numbers, serum albumin concentration and pH, while citrate concentration was lower in comparison to uninfused glands.
• 3.3. Levels of lactose from both infused and uninfused udder halves were normal during the first week of lactation.
• 4.4. No differences were observed in degree of alveolar development in tissue samples collected prior to treatment.
• 5.5. Light and electron microscopy suggested that colchicine-treated udder halves consisted predominantly of undifferentiated mammary secretory cells, while uninfused udder halves appeared more cytologically differentiated.
• 6.6. Results demonstrated that intramammary colchicine infusion at parturition temporarily altered milk composition and inhibited mammary cellular differentiation.

     

Characterization of sheep hepatocytes in primary culture

• 1.1. Primary cultures of isolated sheep hepatocytes were used to characterize metabolic functions of liver: gluconeogenesis, ureagenesis and protein synthesis. The rates of all three metabolic activities were linear over a 20 hr culture period.
• 2.2. Hepatocytes in the presence of glucagon increased the synthesis of urea by approx 30% (P < 0.05) and increased release of glucose into the medium by 60% (P < 0.05).
• 3.3. In the absence of insulin, significantly more (35%; P < 0.05) glucose was released in the medium than in the presence of insulin.
• 4.4. Results help evaluate the primary culture of sheep hepatocytes as an appropriate experimental model to study nutritional and hormonal regulation of liver in the ruminant species.

     

Effects of starvation and a carbohydrate-rich diet on glycogen metabolism in a gastropod mollusc,Megalobulimus oblongus

• 1.1. Administration of a carbohydrate-rich diet increased haemolymph glucose levels and glycogen concentration in hepatopancreas, mantle and muscle.
• 2.2. Glycogen concentration in tissues decreases after 2 weeks of starvation and haemolymph glucose levels did not change significantly.
• 3.3. However, starvation did not induce a decrease in the intrinsic synthetic capacity in tissues.
• 4.4. Glycogen synthesis in tissues from animals fed with lettuce or a carbohydrate-rich diet, increases with increasing glucose concentration in the media.
• 5.5. However, in mantle slices from snails adapted on a carbohydrate-rich diet, the glycogen synthetic capacity was lower than in slices from snails fed with lettuce.

     

Effects of protein synthesis inhibitors on A- and L-site amino acid incorporation by bullfrog tadpole liver and tail fin cells

• 1.1. Cycloheximide and puromycin inhibited leucine transport and incorporation into isolated bullfrog tadpole tail and hepatic cells.
• 2.2. However, high concentrations of these 2 inhibitors did not affect alanine incorporation appreciably in either tissue.
• 3.3. NEM and DNP inhibited leucine and alanine incorporation in both cell types, but at different concentrations.
• 4.4. NEM stimulated leucine transport only in hepatocytes; alanine transport was inhibited by NEM in tail fin cells.
• 5.5. The results suggest different mechanisms of transport and protein synthesis for the 2 types of amino acids by tadpole liver and tail fin cells.

     

Alkaline phosphatase from Basidiobolus haptosporosus: Comparison with alkaline phosphatases from rainbow lizard and man

• 1.1. DEAE-cellulose chromatography of mycelial alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) from Basidiobolus haptosporosus, produced three iso-enzymes “A”, “B” and “C”.
• 2.2. Fraction “A” was further characterized and showed maximum activity at pH 10 in 0.1 M sodium carbonate-bicarbonate buffer.
• 3.3. The enzyme was stimulated by Mg²⁺, Co²⁺ and Mn²⁺ and inactivated by Zn²⁺, Cu²⁺, EDTA, citrate and tartrate.
• 4.4. Phosphate ions inhibited it competitively, phenylalanine uncompetitively and urea noncompetitively.
• 5.5. It was heat stable for 60 min at 37°C but labile above 55°C.
• 6.6. Its K_m with p-nitrophenylphosphate was 0.5 mM; its estimated molecular weight was 160,000.
• 7.7. The results are compared with the properties of alkaline phosphatases from the rainbow lizard and man and discussed in terms of a triadic association between the fungus, the lizard and man.

     

Gluconeogenesis in hepatocytes determined with [2-13C] acetate and quantitative 13C NMR spectroscopy

• 1.1. In the present study the major metabolic pathways of glucose metabolism were determined in isolated liver cells using [2-¹³C]acetate and ¹³C magnetic resonance spectroscopy.
• 2.2. The relative reaction rates of glucose synthesis to the TCA cycle were determined from the ¹³C distribution in glucose where the overall ¹³C enrichment of glucose was 6.41 ± 1.94% (mean ± SD; n = 6) and the mean ¹³C enrichment of C1, C2, C5, C6 to C3, C4 was 2.63 ± 0.30.
• 3.3. Since the distribution of tracer in glucose is a function of the relative entry rates of pyruvate to acetyl-CoA into the oxaloacetate pool this was calculated to be 0.32 ± 0.15 and the factor for carbon exchange (1/P) between the gluconeogenic pathway and the TCA cycle was calculated to be 1.03 ± 0.20.
• 4.4. With this carbon exchange factor and the approximated ¹³C enrichment of acetyl-CoA the intramitochondrial ¹³C enrichment of phosphoenolpyruvate was calculated and the “true” rate of hepatic gluconeogenesis from phosphoenolpyruvate estimated.
• 5.5. Since acetate was metabolized solely in liver cells the ¹³C enrichment of acetyl-CoA could be approximated from that of 3-hydroxybutyrate.
• 6.6. The carbon 13 enrichment of 3-hydroxybutyrate and phosphoenolpyruvate was 5.89 ± 0.90% and 5.96 ± 1.67%, respectively.
• 7.7. The per cent gluconeogenesis from phosphoenolpyruvate calculated as the ratio of the ¹³C enrichment of glucose to that of 3-hydroxybutyrate times 1/P was 107 ± 8%.
• 8.8. In this study the validity of assessing isotopic exchange at oxaloacetate as suggested by Katz [Katz J. (1985) Am. J. Physiol.248, R391–R399] when interpretation of the data are not obscured by pseudoketogenesis.
• 9.9. Magnetic resonance spectroscopy provides direct information about intramolecular tracer distribution by which flux rates in major metabolic pathways are derived.

     

Electrical transport characteristics of Aplysia californica intestinal epithelium

• 1.1. Replacing chloride (Cl⁻) with sulfate (SO₄²⁻) in the bathing medium drastically reduced the mucosal membrane potential difference (ψ_m).
• 2.2. The voltage divider ratio was significantly greater than one.
• 3.3. Mucosal ^d-glucose decreased the input resistance of the intestinal epithelium.
• 4.4. Addition of furosemide to the mucosal bathing medium inhibited transepithelial potential difference and short-circuit current.
• 5.5. Addition of SITS to the mucosal bathing medium partially inhibited transepithelial potential difference and short-circuit current.
• 6.6. Diffusion potentials in the intestinal epithelium were symmetrical.

     

Evidence that protein kinase C is involved in regulating glucose transport in the adipocyte

• 1.1. The role of protein kinase C in the mechanism of stimulation of glucose transport in rat adipocytes was investigated.
• 2.2. Glucose transprt was stimulated by dioleoylglycerol (DOG), tetradecanoyl phorbol acetate (TPA) and phospholipase C (PLC).
• 3.3. Agents that inhibit protein kinase C (polymyxin B, gossypol and quercitin) also inhibited glucose transport that had been stimulated by DOG, TPA, PLC and insulin.