Regulation of glutamine catabolism in the perfused guinea-pig liver in relation to ureogenesis and gluconeogenesis

• 1.1. L-Glutamine conversion into ammonia, urea and glucose by the perfused liver of 48 hr starved guinea-pigs was concentration dependent attaining the maximal rate at 4 mM.
• 2.2. The activity of glutaminase I (EC 3.5.12), measured in isolated liver mitochondria was high enough to account for the observed rate of ammonia, urea and glucose formation by the perfused liver. Neither NH4C1 (5 mM) nor aminooxyacetate (0.5 mM) affected the rate of glutamine conversion into glutamate by isolated liver mitochondria.
• 3.3. Gluconeogenesis and ureogenesis from glutamine was inhibited by octanoate, Dt-3-hydroxybutyrate, aminooxyacetate, ethanol and p-hydroxyphenylpyruvate while ammonia formation was stimulated by aminooxyacetate. 2,4-Dinitrophenol stimulated the rate of the formation of all three metabolites from glutamine.
• 4.4. The major changes induced by aminooxyacetate, as determined in livers perfused with glutamine and stopped by freeze-clamping technique, consisted in a decrease in the content of ATP, aspartate and malate and in a slight increase in the content of glutamate.
• 5.5. Glutamine is an effective precursor of phosphoenolpyruvate in isolated liver mitochondria. Its formation was inhibited by octanoate and by DL-3-hydroxybutyrate.
• 6.6. The data are discussed in terms of regulation of glutamine catabolism in liver with emphasis on ureogenesis and gluconeogenesis.

     

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

     

Mitochondria from the ventricle of the marine clam,Mercenaria mercenaria: Substrate preferences and effects of pH and salt concentration on proline oxidation

• 1.1. Mitochondria with high respiratory control ratios (RCR) have been isolated from the ventricle of the marine clam Mercenaria mercenaria.
• 2.2. Proline is the preferred substrate of the mitochondria of the ventricle based on state 3 rates.
• 3.3. Pyruvate, ornithine and succinate are oxidized at rates 3/4 that of proline.
• 4.4. α-Glycerophosphate was oxidized at rates 1/2 that of proline.
• 5.5. The pH optimum for proline oxidation lies between 6.5 and 7.5 based on RCR and ADP/O and between 7.0 and 7.4 based on state 3 rates.
• 6.6. KCl concentrations between 250 and 450 mM gave optimal values for the oxidation of proline based on RCR and state 3 rates.
• 7.7. KCl concentration had little effect on ADP/O between 100 and 850 mM.

     

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.

     

Effects of a purine inhibitor,allopurinol, on urate metabolism in the german cockroach,Blattella germanica L. (Dictyoptera: Blattellidae)

• 1.1. Adult male and female cockroaches (Blattella germanica) were maintained on a positive nitrogen balance diet (66% protein) containing various levels of allopurinol (0–3%) to determine the effects of allopurinol on urate synthesis and storage.
• 2.2. Each insect was injected with [¹⁴C]hypoxanthine and after 1 week was analyzed for whole-body hypoxanthine, xanthine and urate radiolabel.
• 3.3. There was a general trend of decreased whole-body radiolabel retention, radiolabeled body urates and total-body urate content in both sexes with increasing amounts of dietary allopurinol.
• 4.4. Virgin female adults were allowed to feed on diets containing 0, 25 and 66% protein plus 0.1% allopurinol and were injected with [¹⁴C]xanthine.
• 5.5. After 1 week radiolabel content in the whole-body xanthine and urate pools was determined.
• 6.6. Females on the 0% protein diets contained less radiolabel in the whole-body and body urates than those on either 25 or 66% protein diets.

     

The effect of water loss upon the urate,urea and ammonia content of the egg of the Japanese quail Coturnix coturnix japonica

• 1.1. The urate, urea and ammonia content of the whole egg of the Japanese quail was measured in late incubation in eggs subject to different rates of water loss.
• 2.2. High rates of water loss substantially increased egg urate content, but had little or no effect on urea or ammonia content.
• 3.3. Allopurinol, an inhibitor of urate synthesis, reduced egg urate content to low levels, but produced no effect on urea content, and a small reduction in ammonia content.
• 4.4. The urea concentration of the embryo was lower than in allantoic fluid.
• 5.5. It is concluded that urate production by the avian embryo is primarily concerned with the modification of allantoic fluid composition.

     

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.

     

Properties and distribution of Mg2+-HCO−3-ATPase in brush border membranes isolated from rat small intestine

• 1.1. The small intestine was cut into seven segments and properties and distribution of brush border Mg²⁺-HCO⁻₃-ATPase activity in each segment were examined.
• 2.2. The optimal Mg²⁺ concentration was 1.0 mM.
• 3.3. The optimal HCO⁻₃ concentration was 100 mM in the first (duodenal), 50 mM in the 3rd and 40 mM in the 5th segment, respectively.
• 4.4. The optimal pH value was about 9.0.
• 5.5. l-phenylalanine (above 1 mM) and SCN⁻ (above 50 mM) significantly inhibited both Mg²⁺- and Mg²⁺-HCO⁻₃-ATPase activity.
• 6.6. The enzyme activity was found to be highest in the duodenal segment and then gradually decreased in consecutive segments as well as β-glycerophosphatase, Na⁺-K⁺-ATPase and supernatant carbonic anhydrase.
• 7.7. The functional significance of this ATPase and the relationship with carbonic anhydrase was discussed.

     

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.

     

A new glutamate dehydrogenase from Halobacterium halobium with different coenzyme specificity

• 1.1. Halobacterium halobium has two chromatographically distinct forms of glutamate dehydrogenase which differ in their thermolability and other properties. One glutamate dehydrogenase utilizes NAD, the other NADP as a coenzyme.
• 2.2. The NADP-specific glutamate dehydrogenase (EC 1.4.1.4) was purified 65-fold from crude extracts of H. halobium.
• 3.3. The Michaelis constants for 2-oxoglutarate (13.3 mM), ammonium (3.1 mM) and NADPH (0.077 mM) indicate that the enzyme catalyzes in vivo the formation of glutamate from ammonium and 2-oxoglutarate.
• 4.4. The amination of 2-oxoglutarate by NADP-specific glutamate dehydrogenase is optimal at the pH value of 8.0–8.5. The optimal NaCl or KCl concentration for the reaction is 1.6 M.
• 5.5. None of the several metabolites tested for a possible role in the regulation of glutamate dehydrogenase activity appeared to exert an appreciable influence on the enzyme.
• 6.6. NAD- and NADP-dependent glutamate dehydrogenases from H. halobium showed apparent molecular weights of 148,000 and 215,000 respectively.

     

Regulation of spermidine transport in l1210 cells

• 1.1. 1 mM 2-amino isobutyric add (AIB), glutamine or asparagine when preincubated for 3 hr with L1210 cells promoted a marked increase in the rate of spermidine uptake.
• 2.2. Cycloheximide also increased the transport rate and completely prevented the increase due to AIB.
• 3.3. Trifluoperazine and iso-H7 inhibited the uptake of spermidine, much less the uptake of AIB.
• 4.4. Adenosine promoted an increase in the uptake of AIB, a decrease in that of spermidine.
• 5.5. Hypotonic stress also increased the rate of spermidine transport. This modification was only partially prevented by cycloheximide.
• 6.6. Okadaic arid had no effect on this increase, whereas it prevented the increase of ODC activity.

     

Net glucose production from acetone in isolated murine hepatocytes. The effect of different pretreatments of mice

• 1.1. To evaluate the condition under which net glucose production from acetone, added as sole substrate, occurs different pretreatments of mice, in combination with starvation, were used; (i) acetone pretreatment (acetone is a known inducer of cytochrome P-450 isozymes involved in this pathway), (ii) fructose pretreatment (to induce NADPH + H⁺ generating enzymes) or (iii) their combination.
• 2.2. There was net glucose formation from acetone only in that case, when the cells were prepared from 48 hr fasted animals pretreated with both acetone and fructose. However, using 2-¹⁴C-acetone, incorporation of ¹⁴C-carbon into glucose could be detected in all the cases and, at the same time, acetone was without any effect on protein synthesis.
• 3.3. The addition of acetone increased gluconeogenesis from alanine in almost all the cases. The only exception from this general rule was that the case, when hepatocytes were prepared from acetone pretreated 48 hr starved mice where, instead of the elevation of glucose formation, a decrease of that was caused by acetone.
• 4.4. Acetone decreased ¹⁴C-carbon incorporation into glucose from ¹⁴C-(U)-alanine added at saturating concentration in hepatocytes prepared from starved mice.
• 5.5. Similarly to acetone there was no net glucose formation from acetol either when added alone, however, it enhanced gluconeogenesis from alanine at non-saturating concentrations of the amino acid.
• 6.6. Methylglyoxal proved gluconeogenic in all the cases.
• 7.7. It is concluded that net glucose formation from acetone as sole substrate occurs only under those conditions which are far from a physiological situation, however, when gluconeogenesis from another substrate takes place, acetone can contribute to net glucose formation in hepatocytes prepared from fasted mice.

     

Effect of aminooxyacetate and α-ketoglutarate on glutamate deamination by rat kidney mitochondria

• 1.1. Glutamate dehydrogenase flux by rat kidney mitochondria incubated with 1 mM glutamine plus 2–3 mM glutamate was stimulated by aminooxyacetate. This effect was inhibited by α-ketoglutarate.
• 2.2. Studies with intact mitochondria and mitochondrial sonicates revealed a linear inverse relationship between glutamate deamination and α-ketoglutarate levels.
• 3.3. The data revealed that α-ketoglutarate is a competitive inhibitor of glutamate dehydrogenase with an apparent K_i of 0.6mM.
• 4.4. The data suggest that aminooxyacetate stimulates glutamate deamination by a mechanism mediated by α-ketoglutarate.

     

The effects of duodenal glucose infusion on some hepatic enzyme activities in sheep

• 1.1. Two experiments were performed to examine the effects of duodenal glucose infusion on hepatic enzyme activities in sheep.
• 2.2. Glucose infusion significantly increased the specific activities of phosphofructokinase, pyruvate kinase and 6-phosphogluconate dehydrogenase and significantly reduced the specific activity of glucose-6-phosphatase suggesting that the pathways of glucose breakdown are increased, and gluconeogenesis decreased, in glucose-infused animals.
• 3.3. These results are discussed in relation to the effects of diet on liver metabolism in sheep.

     

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.

     

Evidence for hyporegulation in the calanoid copepod,Acartia tonsa

• 1.1. The euryaline calanoid copepod, Acartia tonsa, maintains haemolymph Na below that of the external medium in salinities above 34^o_oo (475 mM Na).
• 2.2. The measured transepithelial electrical potential. −9.97 ± 1.0 mV, indicates that Na is regulated out of electrochemical equilibrium.
• 3.3. Water osmotically lost in hyporegulation is replaced by Na-dependent absorption by the gut.
• 4.4. High osmotic water permeability is evidenced by the fact that with an increase in external salinity from 475 mM Na to 580 mM Na the copepod's drinking rate nearly doubles.
• 5.5. Sodium efflux measurements indicate that ionic permeability is much lower than other hyporegulating crustaceans.
• 6.6. The energetic advantage of hyporegulation in this species is considered.

     

Basis for hyperexcitation symptom caused by high doses of chlordimeform in the American cockroach

• 1.1. Exposure of isolated ventral nerve cords of the American cockroach to chlordimeform was followed by spontaneous and stimulus-induced hyperactivity which different from that induced by dieldrin in the distribution of spike amplitudes, the delay between stimulus and response and the stimulus threshold to induce hyperactivity.
• 2.2. Calcium (20 mM) abolished the impulse-evoked hyper-response of the nerve to chlordimeform but not to dieldrin.
• 3.3. Magnesium (40 mM) abolished the impulse-evoked hyper-responce of the nerve to dieldrin but not to chlordimeform.
• 4.4. Treatment of the insects with reserpine prior to dissection had no effect on the subsequent hyper-response of the isolated nerve to chlordimeform.
• 5.5. The observed action of chlordimeform is probably axonal, and is unrelated to its known aminergic effects.

     

On the synthesis and incorporation of catalase and urate oxidase into the peroxisomes of mouse liver

• 1.1. The processes associated with the biogenesis of peroxisomes in mouse liver have been studied by following the incorporation of radiolabelled leucine into major enzymic components of this organelle.
• 2.2. Maximal incorporation of label into peroxisomal catalase and urate oxidase occurred within 2 hr, with the urate oxidase being labelled before catalase, but subsequent to the incorporation of phospholipid into this organelle.
• 3.3. Subsequently, immunoprecipitation of catalase from the large granular fraction of mouse liver was shown to result in the isolation of a catalase molecule which had lost a peptide of approx. 2000 dalton from each subunit by comparison with the newly-synthesized enzyme.
• 4.4. It was observed that the modification of catalase was obviated by the presence of leupeptin and iodoacetamide and this information has enabled the purification of both modified and unmodified forms of the enzyme.
• 5.5. The possible significance of these data has been discussed and the major features incorporated into a working model of peroxisomal biogenesis.

     

A comparative study of nuclear and chloroplast DNA dependent RNA polymerases from wheat leaves

• 1.1. Three DNA dependent RNA polymerases have been purified from chromatin and chloroplast fractions of wheat leaves.
• 2.2. The purified enzymes were completely dependent on exogenous DNA after purification by glycerol gradient, DEAE-Sephadex and phosphocellulose chromatography.
• 3.3. The nuclear enzymes, I and II, showed a strong preference for denatured nuclear DNA, whereas the chloroplast enzyme preferred denatured chloroplast DNA.
• 4.4. The three enzymes require either Mg²⁺ or Mn²⁺ for activity.
• 5.5. α-amanitin specifically inhibited RNA polymerase II but has no effect on polymerase I and chloroplast polymerase.
• 6.6. Enzyme I is most active at very low ionic strength (0.10 mM KC1), whereas enzyme II and chloroplast enzyme show maximum activity at 150mM and 50 mM KC1 respectively.