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.

     

Intramitochondrial reductive carboxylation of 2-oxoglutarate in adipose tissue and its contribution to fatty acid synthesis

• 1.1. The reductive carboxylation of 2-oxoglutarate was found to proceed in mitochondria of rat epididymal fat pads and rabbit perirenal adipose tissue at a rate similar to that in liver mitochondria.
• 2.2. In rat fat pads the incorporation of ¹⁴C from [5-¹⁴C]2-oxoglutarate into fatty acids via the carboxylation was suppressed by butylmalonate by 30%.
• 3.3. 2-Oxoglutarate and glutamate stimulated the incorporation into fatty acids of ¹⁴C from [2-¹⁴C]acetate in rat fat pads with the simultaneous reduction of tissue NADP. These effects persisted after inhibition of succinate dehydrogenase by malonate.
• 4.4. It is concluded that in adipose tissue 2-oxoglutarate carboxylation proceeds in both the cytoplasm and mitochondria. Therefore, it can supply carbon atoms as well as NADPH for fatty acid synthesis.

     

Contrast in adenine uptake by chicken and rabbit erythrocytes in vitro

• 1.1. Relative to rabbit erythrocytes, chicken red blood cells exhibit a much greater capacity to utilize [³H]adenine for nucleotide synthesis in vitro, even at 5°C and in the absence of added inorganic phosphate.
• 2.2. This difference is largely due to a higher concentration of phosphoribosylpyrophosphate and greater activity of adenine phosphoribosyltransferase in the avian cells. lli]3. The capacity of avian erythrocytes for utilization of guanine and hypoxanthine is several fold less than that of adenine.
• 3.4. The data are consistent with lower activity for hypoxanthine/guanine phosphoribosyltransferase than for adenine phosphoribosyltransferase in intact chicken erythrocytes.
• 4.5. The results indicate that reutilization of adenine by chicken erythrocytes may be physiologically significant.

     

Amino acid synthesis during hyperosmotic stress in penaeus aztecus postlarvae

• 1.1. Glycine, proline, and taurine are the quantitatively most important amino acid osmolytes in Penaeus aztecus postlarvae.
• 2.2. Taurine dominates the amino acid pool in low salinity, while proline dominates the amino acid pool at higher salinities.
• 3.3. Although not major contributors to the pool, glutamate and alanine are constitutively synthesized from [¹⁴C]glucose and [¹⁴C]glutamate under constant salinity and under hyperosmotic stress treatments.
• 4.4. Proline synthesis from [¹⁴C]-precursors is apparent under constant high (but not low) salinity and is significantly induced by hyperosmotic stress.
• 5.5. No appreciable glycine synthesis was observed from [¹⁴C]glucose or [¹⁴C]glutamate under any experimental conditions.

     

Synthesis of reserved polysaccharide from wax esters accumulated as the result of anaerobic energy generation in Euglena gracilis returned from anaerobic to aerobic conditions

• 1.1. Euglena gracilis SM-ZK (a non-photosynthetic mutant), cultured in Koren-Hutner medium, containing glucose, malate and glutamate as the main nutrients, were incubated anaerobiosis for 24 hr, and then returned to aerobic conditions. Wax esters, which were synthesized from paramylon (the reserved polysaccharide) for ATP generation under anaerobiosis (wax ester fermentation) were promptly degraded immediately after the cells were replenished with sufficient O₂. A large part (about 70%) of the decomposed wax esters were converted back to paramylon.
• 2.2. When cells were fed with [1–14C]acetate or [U-¹⁴C]acetate immediately after transfer from anaerobic to aerobic conditions, radioactivity incorporated into paramylon in the cells fed with [U-¹⁴C]acetate was about 1.5-times as high as that with [1-¹⁴C]acetate, proposing that glyoxylate cycle participates in the conversion from wax esters to paramylon.
• 3.3. Paramylon synthesis from [1-¹⁴C]acetate was considerably activated by anaerobic preincubation of cells for several hours.
• 4.4. Isocitrate lyase and malate synthase occurred in cells cultured in Koren-Hutner medium, but the activities were obviously lower than those in cells grown on ethanol. These enzymes were not induced by the anaerobic preincubation.

     

Neutral amino acid transport in an androgen-dependent epithelium

• 1.1. Transport of neutral amino acids by the isolated seminal vesicle epithelium of normal and gonadectomized guinea pigs has been investigated by measurement of the uptake of 2-amino[1-¹⁴C]-isobutyric acid and 2-methylamino[1-¹⁴C]isobutyric acid.
• 2.2. The V_max for Na-dependent and -independent transport of both amino acids was reduced by gonadectomy but the general transport characteristics appeared to be unchanged by this treatment.
• 3.3. The most likely explanation of the decreased transport is the loss of transporter molecules associated with the tissue regression that follows rapidly on gonadectomy.

     

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.

     

Age-related changes in total dna and rna and incorporation of uridine and thymidine in rat liver,kidney and spleen

• 1.1. Total content of DNA and RNA in liver, kidney and spleen were measured in young and aged rats. At the same time the incorporation of [¹⁴C]thymidine, a DNA precursor, and [³H]uridine, an RNA precursor, were also determined.
• 2.2. Changes in total organ DNA and RNA correlated with sexual maturation as did incorporation of precursors.
• 3.3. Young animals have more DNA per organ relative to RNA. with kidney and spleen DNA showing a decrease between maturity and senescence.
• 4.4. However, liver RNA increases with age. a change probably due to decreased catabolism of RNA since [³H]uridine uptake decreases.
• 5.5. Liver polyploid differentiation, and [¹⁴C]thymidine and [³H]uridine uptake, are correlated.
• 6.6. In kidney, incorporation of [³H]uridine is inversely related to [¹⁴C]thymidine incorporation.

     

Biosynthesis of lipid-linked oligosaccharides in embryonic liver. Glucosylation of mannose containing dolichyl diphosphate oligosaccharide

• 1.1. A maximum rate of dolichyl phosphate [¹⁴C]glucose synthesis from 55-day embryos was achieved at 16nM concentration of exogenous dolichyl phosphate and exceeded about 3 times that without addition of dolichyl phosphate.
• 2.2. The highest values of [¹⁴C]glucose incorporation from UDP-[¹⁴C]glucose into dolichyl phosphate [¹⁴C]glucose, dolichyl diphosphate [¹⁴C]Glc-oligosaccharides and proteins were reached at 5 min time point of incubation of liver microsomes both from embryos and sows.
• 3.3. The radioactive incorporation into proteins was about 7-fold higher in liver microsomes from sows compared to that from embryos, probably due to the greater content of acceptor proteins in microsomes from sows.
• 4.4. The enzymatic transfer of Glc₃-oligosaccharide from a lipid carrier to endogenous protein acceptor in microsomes from pig embryonic and adult livers was considerably faster than the removal of glucose residues during the initial stages of processing of protein-bound oligosaccharides.
• 5.5. One labelled compound was discovered in the Chcl₃-Ch₃Oh-H₂O (1:1:0.3, by vol) extract after incubation of liver microsomes from embryos and sows with UDP-[¹⁴C]glucose. On the basis of its mobility on the chromatogram it appears to be GlcNAc₂Man₉Glc₃.

     

Metabolic utilization of leucine in a fat and a lean line of chicken

• 1.1. In vivo incorporation into body lipids and breast muscle proteins from l-[U-¹⁴C]leucine was studied in genetically lean or fat male chickens, fed or starved, 1 or 24 hr after intraperitoneal injection.
• 2.2. Lipogensis and portein synthesis from labelled leucine were significantly higher in fat chickens than in lean birds, particularly in those in the fed state.
• 3.3. Radioactivity in the free amino acid pool was greater in fat birds irrespective of the nutritional state.
• 4.4. However, utilization of injected l-[U-¹⁴C]leucine for lipogenesis was no more than 2%.

     

Absorption and blood clearance of labelled carotenoids ([14C]astaxanthin, [3H]Canthaxanthin and [3H]zeaxanthin) in mature female rainbow trout (Oncorhynchus mykiss)

• 1.1. Using one force-fed meal, eight mature female rainbow trout received [¹⁴C]astaxanthin ([¹⁴C]Ax) with [³H]canthaxanthin ([³H]Cx; N = 3) or with [³H]zeaxanthin ([³H]Zx; N = 5).
• 2.2. Approximately 200 μl of blood were collected via caudal puncture every 24 hr for 4 days. After 96 hr, the fish were killed and pyloric caeca (P.C.) from the duodenal intestine (D.I.) section, ileal intestine (I.I.), and posterior intestine (P.I.) were dissected out.
• 3.3. In the blood, Ax levels were higher than Cx followed by Zx levels.
• 4.4. This corresponds to their respective absorption by the trout as was confirmed by their relative concentrations in P.C., I.I. and P.I.
• 5.5. However, blood clearance was similar for all three compounds. [¹⁴C]Phoenicoxanthin ([¹⁴C]Px) was detected as a reduced metabolite of [¹⁴C]Ax in all gut sections.

     

Effect of inorganic phosphate on synthesis of 5-phosphoribosyl-1-pyrophosphate in cultured plant cells

• 1.1. Inorganic phosphate (Pi) was absorbed rapidly by suspension-cultured cells of Catharanthus roseus which had previously been cultured in Pi-free Murashige Skoog medium.
• 2.2. The intracellular levels of ATP, ADP and 5-phosphoribosyl-l-pyrophosphate (PRPP) increased markedly during the 24 hr which followed the addition of Pi (1.25mM).
• 3.3. Availability of PRPP in vivo, estimated by the measurement of nucleotide synthesis from [8-¹⁴C]adenine, was also increased by addition of Pi.
• 4.4. Only a 20% increase in the maximum catalytic activity of PRPP synthetase was observed in extracts of cells, prepared 24 hr after addition of Pi.
• 5.5. In contrast to results for mammalian PRPP synthetase, the activity of PRPP synthetase, partially purified from Catharanthus roseus, was inhibited by concentration of Pi greater than 5mM.
• 6.6. The mechanisms involved in the increased availability of PRPP and the synthesis of adenine nucleotides in the plant cells cultured in Pi-containing medium are discussed.

     

Light-induced transient increase of the activity of topoisomerase I in plasmodia of Physarum polycephalum

• 1.1. Activity of topoisomerase I and incorporation of [³H]uridine and [¹⁴C]thymidine were monitored during light-induced sporulation of the slime mold Physarum polycephalun.
• 2.2. A 4-fold transient increase of topoisomerase I activity but not of [³H]uridine or [¹⁴C]thymidine incorporation was observed after 42 hr of illumination with 6 hr impulses.
• 3.3. The activity of topoisomerase I did not increase in the absence of light impulses. However, ca 5-fold increase of the activity was observed in dark when 100 μ M dibutyryl-cAMP was administered 12 hr before harvesting of plasmodia.
• 4.4. Fluorodeoxyuridine and cycloheximide administered 36 hr after starting of the illumination cancelled the increase of the activity of topoisomerase I.
• 5.5. After 7 days of the illumination, when fruiting bodies appeared, the activity of topoisomerase I dropped to about 15% of the initial value.

     

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.

     

In vitro absorption of [14C]leucine during inflammation and the effect of antiinflammatory drugs in the jejunum of rats

• 1.1. Inflammation was induced in the hindlegs of rats by formalin injection and the in vitro absorption of [¹⁴C]leucine was studied.
• 2.2. Treatment of rats with formalin caused a reduction in the in vitro absorption of leucine from the mucosa of jejunum.
• 3.3. Oral administration of oxyphenbutazone or a herbal anti-inflammatory drug (Withania somnifera) prior to formalin injection resulted in no alteration in the jejunal absorption of [¹⁴C]leucine.

     

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.

     

The effect of prolonged fasting on total lipid synthesis and enzyme activities in the liver of the European eel (Anguilla anguilla)

• 1.1. The extent of fatty acid synthesis from [1-¹⁴C]acetate in liver slices was reduced 6-fold when eels were fasted for 1–7 weeks and 20-fold when fasted for 39 weeks; thereafter hepatic lipogenesis seemed to remain constant for up to 95 weeks of fasting.
• 2.2. After a 1–3 week fast some hepatic enzyme activities were reduced (acetyl-CoA carboxylase decreased 2-fold and fatty acid synthetase declined 5-fold), while others remained unchanged (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, α-glycerol phosphate dehydrogenase as well as malic enzyme and ATP-citrate lyase).
• 3.3. The optimum temperature for measuring both total lipid synthesis and lipogenic enzyme activity in eel liver was found to be 30°C.

     

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.

     

Absorption of glucose,glycine and palmitic acid by isolated midgut and hindgut from crickets

• 1.1. In vitro experiments indicated that midgut and hindgut anterior to the Malpighian tubules are important in absorption and processing of products of digestion in crickets.
• 2.2. Isolated hindguts from crickets (Gryllus assimilis, G. rubens and Scapteriscus acletus) absorbed and released into the incubation medium 20–30% of a load of [¹⁴C]glucose and 29–31% of a load of[¹⁴C]glycine.
• 3.3. Isolated midguts from the same crickets absorbed and released into the incubation medium 30–50% of the glucose and 43–52% of the glycine load.
• 4.4. Radiolabelled palmitate was absorbed into epithelial cells of isolated mid- and hindguts, but little was transported and released into the incubation medium.