In vitro substrate utilization for lipid synthesis in liver explants from hyperthyroid chickens

• 1.1. Indian River male broiler chickens growing from 7 to 28 days of age were fed diets containing 12, 18, 24 and 30% protein + 0 or 1 mg triiodothyronine (T₃)/kg of diet to study energetic costs of lipogenesis and the use of various substrates for in vitro lipogenesis.
• 2.2. De novo lipid and CO₂ production were determined in the presence of [1-¹⁴C]pyruvate, [2-¹⁴q]pyruvate, [3-¹⁴C]pyruvate, [2-¹⁴C]acetate and [U-¹⁴C]alanine.
• 3.3. Oxygen consumption was determined in mitochondrial preparations to estimate the energetic costs in expiants synthesizing lipid.
• 4.4. Radiolabeled CO₂ derived from [1-¹⁴C]pyruvate was used as an estimate of coenzyme A availability in liver expiants. Lipids derived from [2-¹⁴C]pyruvate, [2-¹⁴C]acetate and [U-¹⁴C]alanine estimate relative substrate efficiency.
• 5.5. Labeled CO₂ production from [1-¹⁴C]pyruvate was greatest in that group fed a 12% protein diet and least in the group fed a 30% protein diet.
• 6.6. In addition, T₃ increased CO₂ production from [1-¹⁴C]pyruvate.
• 7.7. The production of ¹⁴CO₂ from the second carbon of pyruvate or acetate was increased by T₃.
• 8.8. The low-protein diet (12% protein) increased (P <0.05) lipogenesis.
• 9.9. Adding T₃ to the diets decreased carbon flux into lipid from all substrates, but increased CO₂ production from all substrates without changing stage 3 and 4 respiration rates in mitochondrial preparations.
• 10.10. These observations imply that coenzyme A availability may have regulated de novo lipogenesis in the present study.
• 11.11. It was also concluded that previously noted effects of T₃ on intermediary metabolism may involve metabolic pathways that do not involve changes in mitochondrial function.

     

Cooperative atp inhibition and fructose-1,6-biphosphate activation of rat liver pyruvate kinase

• 1.1. For the determination of relationship between FDP and ATP in the rat liver pyruvate kinase regulation, kinelic studies have been carried out at several ATP and FDP concentrations.
• 2.2. The results obtained on FDP activation show a great cooperativity for FDP saturation with a Hill coefficient of h = 2.79.
• 3.3. Kinetic studies on ATP inhibition also show a great cooperativity for ATP saturation (h = 2.84) at high FDP concentrations.
• 4.4. These results may contribute to explain the regulation of rat liver pyruvate kinase accounting for the activity of this enzyme at high FDP concentrations modulated by small changes in ATP concentrations.

     

Possible involvement of adenylylation in the modification of a 26 kDa protein in rat parotid acinar cells

• 1.1. Adenylylation, a posttranslational modification of proteins, was investigated in saponin-permeabilized acinar cells of the rat parotid gland.
• 2.2. When cells were incubated with [2,8-³H]ATP, several proteins, including a 26 kDa protein in the particulate fraction, were labeled.
• 3.3. Upon incubation of cells with [α-³²P]ATP in the presence of cAMP and 3-isobutyl-lmethylxanthine, ³²P-labeling of the 26 kDa protein was observed.
• 4.4. After treatment with snake venom phosphodiesterase, [³²P]AMP was released from the 26kDa protein. Such release was not observed when cells were labeled with [γ-³²P]ATP.
• 5.5. The ³²P-labeling pattern of proteins with [α-³²P]ATP was clearly different from that with [adenylate-³²P]NAD⁺.
• 6.6. The results suggest that the 26 kDa protein is one of the adenylylation substrates in rat parotid acinar cells.

     

[14C]Formate and [8-14C]Adenine incorporation into purine nucleotides of ischaemic rat liver after re-establishment of blood flow

• 1.1. After restoration of blood flow the incorporation rate of [¹⁴C]formate and [8-¹⁴C]adenine into purine nucleotides of ischaemic rat liver was investigated.
• 2.2. The incorporation rate of [¹⁴C]formate into adenine nucleotides was nearly 5-fold greater in post-ischaemic livers than in controls at 120 min point following blood restoration.
• 3.3. No difference in the rate of [8-¹⁴C]adenine incorporation into adenine nucleotides of post-ischaemic and control rat liver lobes was found.
• 4.4. After re-establishment of blood circulation a predominant contribution of the de novo biosynthetic pathway in the recovery of purine nucleotides of the ischaemic rat liver is emphasized.

     

PLA2 activity in rat liver nuclei

• 1.1. Subcellular fractions of rat liver were assayed for PLA₂ activity.
• 2.2. The PLA₂ assay measures the release of [³ H]oleic acid from phospholipids, using labeled E. coli as substrate.
• 3.3. Nuclear fractions contained PLA₂ activity, which was Ca²⁺ dependent and could not be explained from mitochondrial, microsomal or plasma membrane contamination.
• 4.4. The V_max value of nuclear PLA₂ is 0.30 ± 0.04 pmol oleic acid/min/mg protein; its K_m value is 0.86±0.12μM, similar to that of mitochondrial PLA₂.
• 5.5. We conclude that rat liver nuclei contain PLA₂ activity.

     

Metabolism of benzo[a]pyrene and persistence of DNA adducts in the brown bullhead (Ictalurus nebulosus)

• 1.1. The in vitro metabolism of [³H]benzo[a]pyrene (BP) and [¹⁴C]benzo[a]pyrene-7,8-dihydrodiol (BP-7,8-diol) by liver of brown bullhead (Ictalurus nebulosus) was characterized, as was the formation and persistence of BP-DNA adducts in vivo.
• 2.2. Compared to rat liver microsomes, bullhead liver microsomes produced relatively larger amounts of BP-7,8-diol (predominantly the [−] enantiomer) and smaller amounts of BP-4,5-diol.
• 3.3. BP phase I metabolites were efficiently converted by freshly isolated bullhead hepatocytes to conjugates, predominantly glucuronides.
• 4.4. BP-7,8-diol was metabolized by hepatocytes 4-fold more rapidly than was BP and was converted to approximately equal amounts of glucuronides, glutathione conjugates and sulfates.
• 5.5. BP-DNA adducts formed in bullhead liver with a lag time of several days and maximum adduct formation at 25–30 days. The major adduct was anti-BPDE-deoxyguanosine.

     

The binding and degradation of 125I-labelled insulin by rat kidney brush-border membranes

• 1.1. The interaction of insulin with purified brush-border membranes from rat kidney was studied with the use of [¹²⁵I]insulin.
• 2.2. The specific binding of insulin by brush-borders could be demonstrated, and was time- and temperature-dependent.
• 3.3. [¹²⁵I]insulin was displaced by unlabelled insulin. A₁-B₂₉ dodecoyl insulin and insulin A- and B-chains in proportion to their relative bioactivity.
• 4.4. Brush-border membranes showed high insulin-degrading activity with an apparent K_m of 2.2 μM.
• 5.5. A number of proteinase inhibitors were effective in inhibiting insulin degradation but the greatest degree of inhibition was achieved by the use of thiol-blocking reagents.
• 6.6. No evidence was obtained for the involvement of the enzyme glutathione-insulin transhydrogenase.

     

In vivo and in vitro synthesis of some serum peptides

• 1.1. A high amount of [¹⁴C]leucine is incorporated in vivo into rat serum peptides after 1 hr of isotope administration.
• 2.2. In vitro [¹⁴C]leucine appears in reconstituted blood plasma and in rat liver peptide mixtures. After 4hr of perfusion the radioactivities amount to 2078 ± 913 and 2120 ± 549 cpm/mg of peptides, respectively.
• 3.3. The serum and liver peptides analysed show a great aggregation ability and so it is difficult to decide which of the liver peptides are destined for the serum.

     

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.

     

Purine metabolism in cotyledons and embryonic axes of black gram (Phaseolus mungo L.) Seedlings

• 1.1. The metabolism of purine bases and nucleosides in cotyledons and embryonic axes of black gram (Phaseolus mungo L.) was studied.
• 2.2. A large portion of absorbed [8-¹⁴C]adenine, [8-¹⁴C]guanine and [8-¹⁴C]adenosine was salvaged in nucleotide and nucleic acid synthesis.
• 3.3. Most of the radioactivity of [8-¹⁴C]hypoxanthine and [8-¹⁴C]inosine was incorporated into allantoin and allantoic acid.
• 4.4. Activity of adenine phosphoribosyltransferase in enzyme extracts was much higher than that of hypoxanthme and guanine phosphoribosyltransferase(s).
• 5.5. Apparent activity of adenosine kinase was higher than that of inosine kinase. 6. NAD⁺-dependent xan thine dehydrogenase was detected in both cotyledons and embryonic axes of the seedlings.
• 6.7. The capacity of purine salvage was higher m 24 hr old cotyledons than 24 and 48 hr old embryonic axes. The reverse was observed concerning that of purine degradation.

     

The regulation of pyruvate kinase in the hepatopancreas of Carcinus maenas)

• 1.1. Estimation of the activity of pyruvate kinase and pyruvate carboxylase in the hepatopancreas of Carcinus maenas gave values of 28.5 and 7.4 units/g wet wt tissue respectively.
• 2.2. The concentrations of substrates, products and allosteric effectors of these enzymes in hepatopancreas were measured.
• 3.3. The activities of pyruvate kinase and pyruvate carboxylase were redetermined using the approximate physiological range of substrate and effectors.
• 4.4. Under these conditions the effective activity of pyruvate kinase could be decreased to less than 2 units/g wet wt tissue whereas that for the pyruvate carboxylase was 2.6 units/g wet wt tissue indicating that a net synthesis of phosphoenolpyruvate could occur in vivo.

     

Functional changes of rat brain mitochondrial enzymes induced by monomeric cholesterol

• 1.1. The binding of [¹⁴C]cholesterol into rat brain mitochondrial membranes follows an exponential path described by the general formula y = a.e^bx. [¹⁴C]cholesterol glucoside binding has a sigmoidal character where the “best-fit” curve of this type of binding is the one described by the Hill equation with Hill coefficient h = 2.06. These findings suggest a positive cooperativity in the binding of both compounds into rat brain mitochondrial membranes.
• 2.2. The specific activity of the outer mitochondrial membrane enzyme monoamine oxidase was linearly decreased at different concentration of cholesterol or its glucoside.
• 3.3. The specific activity of the inner mitochondrial membrane enzyme succinate-cytochrome c reductase was linearly decreased, while that of Rotenone-sensitive NADH-cytochrome c reductase was exponentially increased, at different concentrations of cholesterol.
• 4.4. These results are discussed in terms of specific interactions of cholesterol with constituent mitochondrial membrane lipids and their implications for deviations from normal neuronal function.

     

Rat liver endoplasmic reticulum protein kinases

• 1.1. Rat liver microsomal membranes were studied for the presence of protein kinases. Microsomal proteins solubilized with Triton X-100 were analyzed by means of ion exchange chromatography.
• 2.2. Protein kinase activity was detected in the column fractions using specific assays for cAMP-dependent protein kinase, cGMP-dependent protein kinase, protein kinase C, Ca²⁺/calmodulin-dependent protein kinase and casein kinases.
• 3.3. Fractions with protein kinase activity were further analyzed by SDS-polyacrylamide gel electrophoresis.
• 4.4. The results indicate that cAMP-dependent protein kinase type I and II, casein kinases I and II, protein kinase C proenzymes I and II and Ca²⁺ /calmodulin kinase II are associated with the membranes of endoplasmic reticulum (ER).

     

Soluble cyclic amp-dependent protein kinases from chick liver: Effects of NaCl and heat-stable modulator

• 1.1. Two cyclic AMP-dependent protein kinases—Fraction I and II—have been isolated from chick liver soluble preparation on DEAE-cellulose.
• 2.2. Both fractions have an apparent K_m for ATP of 2 × 10⁻⁶M, are stimulated maximally by 5 × 10⁻⁸ M cyclic AMP and phosphorylate mainly basic proteins—histone and protamine.
• 3.3. They exhibit various pH values for optimal activity and show differences with respect to both sensitivity to NaCl and substrate specificity.
• 4.4. The heat-stable protein modulator inhibits the cyclic AMP-dependent protein kinase activity of both fractions, but with cyclic GMP one kinase is stimulated and the other inhibited.
• 5.5. Slight differences in histone triggered holoenzyme dissociation as well as the lack of difference between their ability for subunit reassociation do not allow to classify these isozymes as protein kinases of Type I and II, according to Corbin et al. (1975).

     

Ca2+-dependent stimulation of muscle and liver phosphorylase kinase by heparin

• 1.1. Heparin stimulates the activity of nonactivated and activated skeletal muscle phosphorylase kinase in a Ca²⁺-dependent manner.
• 2.2. The stimulatory effect of heparin on the activity of nonactivated phosphorylase kinase is also expressed in the presence of calmodulin and glycogen. Heparin acted in synergism with glycogen.
• 3.3. Heparin increases the affinity of phosphorylase kinase to Ca²⁺ 5–12 fold depending upon the activation conditions.
• 4.4. Ca²⁺ influences the stimulation of liver phosphorylase kinase by heparin in a similar way.

     

Kidney metabolism of mevalonic acid in neonatal chick

• 1.1. Metabolism of mevalonic acid (MVA) to phosphorylated derivatives has been studied in 1–3 day chicks kidneys. In all experiments only 5-phosphomevalonic acid (MVAP) and 5-pyrophosphomevalonic acid (MVAPP) were observed as phosphorylated derivatives of [2-¹⁴C]MVA.
• 2.2. Properties of mevalonate-activating enzymes from neonatal chick kidney have been established. Supplementation of Mg²⁺ or Mn²⁺ strongly increased the MVA phosphorylation. The enzymes require ATP as nucleotide for optimal activity. ITP can also be utilized less effectively.
• 3.3. Addition of 0.1 mM Hg²⁺ or 0..01 mM p-hydorxymerucibenzoate inhibited MVA phosphorylation, thus suggeting that thiol groups may participate in the active site of mevalonate kinase from chick kidney. However, unlike other mevalonate kinases, the enzyme from chick kidney did not require thiol group protectors as activators.
• 4.4. Phosphomevalonae kinase was completely inactivated after 5–10 min treatment at 50°C, whereas mevalonate kinase was found to retain its activity in the same conditions. Exposure to 65°C for 5–15 min resulted in the inactivation of mevalonate kinase.

     

Generation of C3- and C2-deuterated l-lactic acid by human erythrocytes exposed to d-[1-13C]glucose,d-[2-13c]glucose and d-[6-13C]glucose in the presence of D2O

• 1.1. The generation of C₂- and C₃-deuterated l-lactate was monitored by ¹³C NMR in human erythrocytes exposed to d-[1-¹³glucose, d-[2-¹³C]glucose or d-te-¹³C]glucose and incubated in a medium prepared in D₂O.
• 2.2. The results suggested that the deuteration of the C₁ of d-fructose 6-phosphate in the phosphoglucoisomerase reaction, the deuteration of the C₁ of d-glyceraldehyde-3-phosphate in the sequence of reactions catalyzed by triose phosphate isomerase and aldolase and the deuteration of the C₃ of pyruvate in the reaction catalyzed by pyruvate kinase were all lower than expected from equilibration with D₂O.
• 3.3. Moreover, about 40% of the molecules of pyruvate generated by glycolysis apparently underwent deuteration on their C₃ during interconversion of the 2-keto acid and l-alanine in the reaction catalyzed by glutamate-pyruvate transaminase.
• 4.4. The occurrence of the latter process was also documented in cells exposed to exogenous [3-¹³C]pyruvate.
• 5.5. This methodological approach is proposed to provide a new tool to assess in intact cells the extent of back-and-forth interconversion of selected metabolic intermediates.

     

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.

     

Activation and inhibition of insulin receptor autophosphorylation by trypsin treatment of intact H35 cells

• 1.1. Treatment of intact cultured H35 cells with trypsin (1 mg/ml) for 15 min at low temperature (4°C) or for 30 sec at 37°C causes activation of the insulin receptor subsequently isolated from the cells.
• 2.2. Receptor activation was assessed by increased phosphotyrosine content of the β-subunit of the receptor, and increased autophosphorylation using [³²P]-ATP.
• 3.3. Treatment of the cells for 15 min at 37°C however completely abolished insulin binding and all insulin receptor kinase activity.
• 4.4. These data demonstrate that proteolytic damage of the extracellular domain of the insulin receptor can render the receptor kinase inactive and lead to a cell which is unresponsive to insulin.

     

Selective inhibition by clofenapate of glycerolipid synthesis via the esterification of dihydroxyacetone phosphate in rat liver slices

• 1.1. The route of glycerol incorporation into glycerolipids was measured by incubating rat liver slices with [l-¹⁴C]-glycerol and (2-³H]-glycerol.
• 2.2. Approximately 75% of the incorporation was via the esterification of dihydroxyacetone phosphate with the remainder passing through the glycerol phosphate pathway.
• 3.3. Clofenapate (1.25mM) inhibited lipid synthesis by about 78% and this resulted from a selective inhibition of dihydroxyacetone phosphate incorporation.
• 4.4. A combined inhibition of glycerol phosphate oxidase and dihydroxyacetone phosphate acyltransferase is thought to be responsible for these observations.