Induction of lipid synthesis in mammary organ cultures from mature virgin and pregnant goats.

The hormones needed to induce lipogenesis in mammary organ cultures from mature virgin and pregnant goats were studied. In tissues from both mature virgin goats and goats at week 10 of pregnancy, cultivated in Waymouth medium without hormones, the rate of the incorporation of (1-(14C))-acetate into the lipids was low and decreased throughout culture. In the presence of insulin, the rate of acetate incorporation was maintained at a higher level. Cortisol acted synergistically with insulin, to produce a rate of lipid synthesis higher than that using insulin alone. The further addition of prolactin had little effect on the incorporation of acetate into the lipids of mammary explants from mature virgin goats, but markedly stimulated it in tissue from animals at weeks 9--10 of pregnancy. The maximum increase in the rate of lipid synthesis was achieved in the presence of 0.5 microgram prolactin/ml, whereas with growth hormone 50 microgram/ml was needed for the maximum effect. The initial rate of acetate incorporation into mammary explants from goats at weeks 13 and 18 of pregnancy was high. It was not stimulated by the hormones during culture, however, and decreased more rapidly in the absence of hormones than in their presence. The rate of acetate incorporation into the lipids was in agreement with the histological evaluation of the secretory response of the mammary explants after cultivation. The secretory response to prolactin and the rate of the incorporation of acetate into the lipids were highest in goats at weeks 9--10 of pregnancy while in tissues from goats at weeks 13 and 18 were not stimulated and decreased during culture.     

Effects of a partially purified factor from chick embryos on macromolecular synthesis of embryonic pancreatic epithelia

Essentially normal development of early embryonic pancreatic epithelium occurs only in the presence of mesenchymal tissues (Golosow and Grobstein, 1962), or a particulate fraction (MF) obtained from extracts of chicken embryos (Rutter et al., 1964). We have shown that this fraction also stimulates the incorporation of thymidine-³H into DNA. This stimulatory activity was detected in particulate fractions from homogenates of several mesodermal tissues from rat and chick embryos, as well as in fibroblasts cultured from these tissues, but not in embryonic epithelial tissues. This activity may thus be related to the mesodermal tissue requirement for pancreatic development. MF was solubilized and partially purified from homogenates of chick embryos. It is stable to collagenase, hyaluronidase, and neuraminidase. Activity is lost by heating and by treatment with trypsin. It is presumed, therefore, that the factor is associated with a protein that is not collagen.The effects of the MF upon macromolecular synthesis were tested in pancreatic tissues from 12-day rat embryos. When isolated epithelia were cultured in the absence of mesoderm or MF, the rate of thymidine-³H incorporation into DNA decreased to low levels. The specific activities of DNA polymerase and deoxycytidylate deaminase in epithelial extracts also declined. In contrast, the rate of thymidine-³H incorporation into DNA increased 5- to 8-fold over the initial rates in epithelia cultured with MF. Concurrently DNA polymerase activity in tissue extracts increased by 2- to 3-fold; deoxycytidylate deaminase activity declined slightly.MF also affected RNA and protein synthesis. The rate of leucine-³H incorporation into protein and uridine-¹⁴C incorporation into RNA in isolated pancreatic epithelia was comparable to that of intact rudiments. Cultures in the presence of MF increased these rates severalfold after 20 hr. These results suggest that MF, and by implication, mesoderm, may supply a growth factor for epithelial tissue and thus serves a permissive rather than a determining role in the differentiation process in pancreatic development.     

Enzyme and protein turnover in adipose tissue in pregnancy and lactation

1. The relative rates of synthesis of fatty acid synthetase and the pyruvate dehydrogenase complex were measured in adipose tissue in virgin, late pregnant and early lactating rats after injection of l-[2,3-³H]alanine. The relative rate of synthesis of fatty acid synthetase decreased approximately 4-fold between 2 days prepartum and 2 days postpartum. The relative rate of synthesis of the pyruvate dehydrogenase complex did not change. 2. The fractional rate of total adipose tissue protein synthesis was measured by constant infusion with l-[U-¹⁴C]tyrosine. Total protein synthesis did not differ in virgin and 2-day lactating rats. The half-life of adipose tissue protein in virginn rats determined by decay of ¹⁴C label from protein after injection of NaH¹⁴CO₃ was 86.9 ± 6.7 h. This is in close agreement witht the half-life (82.5 ± 20 h) calculated from the fractional rate of protein synthesis determined by the constant infusion method.     

Lipid biosynthesis in liver slices of the foetal guinea pig.

Lipid synthesis as measured by the incorporation of acetate or 3H2O into slices of foetal liver, is much higher than in slices of adult liver and shows a peak at about two-thirds of gestation. At this time the synthesis from glucose was low and reached a peak 10 days later. The changes in the activity of ATP citrate lyase, which mirrored acetate incorporation, and the effect of glucose and pyruvate on acetate corporation into lipid suggests that some of the lipid synthesis occurs via intramitochondrial acetyl-CoA production from acetate. Despite this, lipid synthesis was not inhibited by (-)-hydroxycitrate. The low rate of synthesis from glucose at two-thirds of gestation is ascribed to the low activity of pyruvate carboxylase at this time and a role for a phosphoenolpyruvate carboxykinase in providing oxaloacetate for lipogenesis is proposed. The activity of fatty acid synthetase broadly agreed with the changes in lipid synthesis, whereas the activity of acetyl-CoA carboxylase was barely sufficient to account for the rates of lipid synthesis in vivo. Acetate and short-chain fatty acids are likely to be the major precursors for lipid synthesis in vivo.     

Studies onC-glycosides in higher plants

The biosynthesis of bergenin and arbutin in the organs ofSaxifraga stolonifera has been studied by tracer techniques. Among the organs tested, the leaves showed the highest incorporation of label fromd-glucose-U-¹⁴C into bergenin and arbutin under continuous light, although every organ is more or less able to synthesize both glucosides. The incorporation rate into bergenin was higher in the young leaves, whereas the synthesis of arbutin became active in the mature leaves. The fact that the addition of unlabeled gallic acid enhanced the incorporation of label into bergenin suggests that gallic acid may be a likely glucosyl acceptor in bergenin biosynthesis.     

Precursor supply and hepatic enzyme activities as regulators of triacylglycerol synthesis in isolated hepatocytes and perfused liver

The relative significance of alterations in precursor supply and enzyme activities for the rate of triacylglycerol synthesis was studied in isolated hepatocytes and perfused livers. Precursor availability was varied in vitro by changing the fatty acid concentration in the incubation medium or adding ethanol to the perfusion medium in order to increase the cellular glycerol 3-phosphate concentration. The rate of glycerolipid synthesis in hepatocytes, measured in terms of the label incorporated into the various lipid classes from tritiated glycerol, was strongly dependent on the fatty acid concentration up to 2 mm of oleate (fatty acid/albumin molar ratio $\text{7}\text{1}$). Ethanol in vitro increased the incorporation of labeled oleate into phosphatidic acid and diacylglycerol in the isolated perfused liver, but its effect on the incorporation into triacylglycerol was small. Ethanol in vitro increased the label incorporation into both diacylglycerol and triacylglycerol in the livers from cortisol-treated rats. Although cortisol treatment increased the soluble phosphatidate phosphohydrolase activity 4.4-fold in the hepatocytes, it had no effect on the rate of triacylglycerol synthesis, whereas fasting increased this rate about 3-fold, although only a moderate concomitant increase in soluble phosphatidate phosphohydrolase activity was observed. Neither cortisol treatment nor fasting affected the microsomal glycerol-3-phoshate acyltransferase activity. The results demonstrate that substrate availability can override enzyme modulations in the regulation of triacylglycerol synthesis and that phosphatidate phosphohydrolase is not the main regulator of triacylglycerol synthesis.     

In vivo protein synthesis from 14C-substrates in porcine tissues during the transition to postnatal development

[2-14C] leucine, [1-14C] alanine, [1-14C] glucose, [1-14C] lactate and [1-14C] pyruvate utilization in the protein synthesis has been studied in vivo at early stages of postnatal development of piglets. It has been established, that during the first 24 hours after birth the protein synthesis intensity, judging by [2-14C] leucine incorporation, in liver, skeletal muscle, duodenal wall and subcutaneous tissue of piglets increases 5, 7, 6.5 and 2.1 times respectively. At the age of 1-2 h the radioactive carbon incorporation from [1-14C] glucose into the brain proteins is more pronounced than into the proteins of liver and skeletal muscle. During the first days of life the intensity of the label incorporation from [1-14C] glucose into liver and skeletal muscle proteins of piglets is enhanced, whereas in brain it remains at the same level. The degree of 14C carbon incorporation from [1-14C]-alanine, [1-14C] pyruvate and [1-14C] lactate into the liver and skeletal muscle proteins of 5-days-old piglets is approximately the same, 14C substrates of protein synthesis in brain and subcutaneous adipose tissue having some peculiarities.     

Differential incorporation of docosahexaenoic and arachidonic acids by the yolk sac membrane of the avian embryo

During avian development, lipoproteins derived from yolk lipid are assembled in the yolk sac membrane (YSM) for secretion into the embryonic circulation. To investigate how yolk polyunsaturated fatty acids, essential for the development of certain tissues, are distributed among the lipid classes of the lipoproteins, pieces of YSM were incubated in vitro with [14C]arachidonic and [14C]docosahexaenoic acids (DHA). There was a marked difference in the partitioning of these two precursors among the lipid classes of the tissue. Of the radioactivity incorporated into total lipid from [14C]-arachidonic acid during 1 h of incubation, 67.3% was esterified as phospholipid and 29.5% as triacylglycerol. In contrast, only 14.6% of the label incorporated from [14C]-DHA was esterified as phospholipid, whereas 73.2% was recovered in triacylglycerol. This pattern of differential partitioning was observed at all time points and across a 20-fold range of fatty acid concentrations. There was no evidence for conversion of the radioactive arachidonic and DHAs to other fatty acids prior to incorporation into tissue lipids. It is suggested that the selective incorporation of yolk-derived DHA into the triacylglycerol of secreted lipoproteins represents part of a mechanism for directing this polyunsaturate to particular embryonic tissues.     

Acetoacetate is a cholesterogenic precursor for myelinating rat brain and spinal cord. Incorporation of label from [3-14C]acetoacetate, [14C]glucose and 3H2O

Rat pups, 3 weeks old, were injected i.p. with combinations of 3H2O and either [3-14C]acetoacetate or [14C]glucose. 3H/14C incorporation ratios were measured in lipid fractions of homogenates and myelin prepared from whole brain and spinal cord. Spinal cord synthesized at least twice as much fatty acids and 3-fold more sterols than whole brain. Both tissues used acetoacetate preferentially for sterol synthesis, whereas label from [14C]glucose was distributed between fatty acids and sterols in the same way as 3H from 3H2O. The relative contributions of acetoacetate to sterol synthesis in whole tissue and in the purified myelin fraction were about the same, both for the cerebrum and for the spinal cord.     

Plasma-membrane phospholipid unsaturation affects expression of the general amino-acid permease in Saccharomyces cerevisiae Y185

Saccharomyces cerevisiae Y185, enriched in linoleyl residues and incubated for up to 4 h in derepression buffer, more rapidly acquired general amino-acid permease (GAP) activity, as measured by the rate of accumulation of L-alanine, compared with organisms enriched in oleyl residues. A GAP-less mutant incubated under the same conditions did not acquire further L-alanine-accumulating ability, irrespective of the nature of the fatty-acyl enrichment. During derepression, KT values for the GAP were virtually identical irrespective of the fatty-acyl enrichment, but Vmax values were greater for linoleyl residue-enriched organisms, particularly after 1 h in derepression buffer. During incubation in derepression buffer, organisms with either fatty-acyl enrichment did not differ in the size of the amino-N pool, the concentration of L-alanine in that pool, rates of protein synthesis and glucose fermentation, or rate and extent of incorporation of label from H2 32PO-4. Under conditions used to measure rates of L-alanine accumulation, organisms with either enrichment showed no evidence of metabolism of accumulated L-alanine.     

Comparative study of RNA metabolism in freshly isolated protoplasts and callus cultures of Parthenocissus tricuspidata crown gall

RNA metabolism was compared in protoplasts and cells of Parthenocissus tricuspidata crown gall callus. A rapid increase in the permeability to precursors during the first three hours following the termination of protoplast isolation was observed. Consequently, RNA synthesis occurred at a higher rate in protoplasts than in whole cells. On the other hand, protoplasts and callus tissues showed similar kinetics of incorporation of precursors into mature rRNA's. Pulse-chase experiments showed the maturation rate to be nearly the same in both cases.     

Altered Synthesis and Composition of Cell Wall of Grape (Vitis vinifera L.) Leaves during Expansion and Growth-Inhibiting Water Deficits

The rate and composition of cell wall polysaccharide synthesisduring development and growth-inhibiting water deficits wereinvestigated in leaves of grape (Vitis vinifera L.). The rateof leaf expansion was monitored as plant water status was manipulatedby modulating the supply of irrigation water to potted plantsover several days. The corresponding wall synthesis was determinedby incubating leaf tissue with [¹⁴C]glucose and quantifyingincorporation into wall components. Samples were obtained fromrapidly expanding and mature leaves before, during, and following(recovery from) moderate water deficits. Uptake was approximately2-fold greater for mature leaf tissue than for rapidly expandingtissue at both high and low water status. In contrast, incorporationinto cell wall polysaccharides was 18 to 41% (under low andhigh water status) of uptake in expanding leaves but less than4% in mature tissue. Incorporation of precursor into wall polysaccharideswas insensitive to plant water status in mature leaves, butwas inhibited to less than 50% of well-watered controls in expandingleaves at low water potential. Incorporation of label into cellulose,uronic acid, and neutral sugar fractions was differentiallyaffected by water deficits, with cellulose synthesis apparentlyexhibiting the greatest sensitivity to low water status. Afterrewatering, growth, as well as uptake and incorporation of labelrecovered, although the latter did not attain prestress rates.The results indicate a high sensitivity of wall polysaccharide(particularly cellulose) synthesis to growth-inhibiting waterdeficits. ¹ Supported by United States Department of Agriculture, CompetitiveResearch grant GAM 8502539. (Received November 15, 1989; Accepted January 17, 1990)     

Tissue kallikrein synthesis and its modification by testosterone or low dietary sodium.

A method has been developed to measure the relative rate of rat tissue kallikrein synthesis which employs a specific antiserum raised against a purified rat urinary kallikrein. Incorporation of [35S]methionine into kallikrein and protein 20 min after intraperitoneal injection was measured in submaxillary gland, pancreas, kidney and descending colon. Kallikrein content was measured with a direct radioimmunoassay, and kallikrein-specific incorporation of [35S]methionine measured after immunoprecipitation. Kallikrein specific radioactivity (c.p.m./mg of enzyme) was about 100-fold greater than that in total protein in both kidney and colon. In contrast, in pancreas the incorporation into the enzyme was only 5-fold higher than into protein, and in submaxillary gland the incorporation was equivalent. Measured as kallikrein-specific radioactivity relative to total protein radioactivity incorporated in 20 min, kallikrein represents 0.18% of total protein synthesis in the kidney, 0.34% in the pancreas, 0.41% in the colon, but 7.29% in the submaxillary gland. Dietary Na+ restriction increased the relative rate of kallikrein synthesis 1.8-fold in the kidney without a comparable effect in submaxillary gland. In contrast, testosterone increased the relative rate of synthesis 2.3-fold in submaxillary gland, but decreased it in kidney. The data show that endogenous kallikrein synthesis differs markedly in various tissues, and that interventions which are known to change kallikrein content or excretion also change the relative rate of enzyme synthesis.     

Utilization of L-alanine and L-glutamine by lactating mammary gland of the rat. A role for L-alanine as a lipogenic precursor.

1. Lactation is associated with an increase in the arterial blood concentration of L-alanine and L-glutamate, but a decrease in that of L-glutamine compared with the corresponding values for virgin rats. 2. Virgin rats fed a 'cafeteria diet' that induces hyperphagia have increased arterial concentrations of L-alanine, L-glutamate and L-glutamine. During lactation L-alanine and L-glutamate concentrations are even higher. 3. The removal of L-alanine is decreased in hepatocytes from lactating rats fed either a chow or cafeteria diet. 4. Measurements of arteriovenous differences across lactating mammary glands indicate that appreciable amounts of L-glutamine and L-alanine are extracted by the gland. 5. A high proportion of the L-alanine metabolized by isolated acini from fed lactating rats is converted into lipid. 6. Metabolism of L-alanine in acini from starved lactating rats is limited by the activity of pyruvate dehydrogenase. 7. It is concluded that L-alanine and certain other amino acids taken up by the gland in excess of the requirements for protein synthesis can be converted into lipid.     

In vivo 13C NMR determines metabolic fluxes and steady state in linseed embryos

The dynamics of developing linseed embryo metabolism was investigated using (13)C-labelling experiments where the real-time kinetics of label incorporation into metabolites was monitored in situ using in vivo NMR. The approach took advantage of the occurrence in this plant tissue of large metabolite pools - such as sucrose or lipids - to provide direct and quantitative measurement of the evolution of the labelling state within central metabolism. As a pre-requisite for the use of steady state flux measurements it was shown that isotopic steady state was reached within 3 h at the level of central intermediates whereas it took a further 6h for the sucrose pool. Complete isotopic and metabolic steady state took 18 h to be reached. The data collected during the transient state where label was equilibrated but the metabolic steady state was incomplete, enabled the rates of lipid and sucrose synthesis to be measured in situ on the same sample. This approach is suitable to get a direct assessment of metabolic time-scales within living plant tissues and provides a valuable complement to steady state flux determinations.     

Leucine incorporation and its potential as a measure of protein synthesis by bacteria in natural aquatic systems

Leucine incorporation was examined as a method for estimating rates of protein synthesis by bacterial assemblages in natural aquatic systems. The proportion of the total bacterial population that took up leucine in three marine environments was high (greater than 50%). Most of the leucine (greater than 90%) taken up was incorporated into protein, and little (less than 20%) was degraded to other amino acids, except in two oligotrophic marine environments. In samples from these two environments, ca. 50% of the leucine incorporated had been degraded to other amino acids, which were subsequently incorporated into protein. The degree of leucine degradation appears to depend on the organic carbon supply, as the proportion of 3H-radioactivity incorporated into protein that was recovered as [3H]leucine after acid hydrolysis increased with the addition of pyruvate to oligotrophic water samples. The addition of extracellular leucine inhibited total incorporation of [14C]pyruvate (a precursor for leucine biosynthesis) into protein. Furthermore, the proportion of [14C]pyruvate incorporation into protein that was recovered as [14C]leucine decreased with the addition of extracellular leucine. These results show that the addition of extracellular leucine inhibits leucine biosynthesis by marine bacterial assemblages. The molar fraction of leucine in a wide variety of proteins is constant, indicating that changes in leucine incorporation rates reflect changes in rates of protein synthesis rather than changes in the leucine content of proteins. The results demonstrate that the incorporation rate of [3H]leucine into a hot trichloroacetic acid-insoluble cell fraction can serve as an index of protein synthesis by bacterial assemblages in aquatic systems.     

Leucine incorporation and its potential as a measure of protein synthesis by bacteria in natural aquatic systems.

Leucine incorporation was examined as a method for estimating rates of protein synthesis by bacterial assemblages in natural aquatic systems. The proportion of the total bacterial population that took up leucine in three marine environments was high (greater than 50%). Most of the leucine (greater than 90%) taken up was incorporated into protein, and little (less than 20%) was degraded to other amino acids, except in two oligotrophic marine environments. In samples from these two environments, ca. 50% of the leucine incorporated had been degraded to other amino acids, which were subsequently incorporated into protein. The degree of leucine degradation appears to depend on the organic carbon supply, as the proportion of 3H-radioactivity incorporated into protein that was recovered as [3H]leucine after acid hydrolysis increased with the addition of pyruvate to oligotrophic water samples. The addition of extracellular leucine inhibited total incorporation of [14C]pyruvate (a precursor for leucine biosynthesis) into protein. Furthermore, the proportion of [14C]pyruvate incorporation into protein that was recovered as [14C]leucine decreased with the addition of extracellular leucine. These results show that the addition of extracellular leucine inhibits leucine biosynthesis by marine bacterial assemblages. The molar fraction of leucine in a wide variety of proteins is constant, indicating that changes in leucine incorporation rates reflect changes in rates of protein synthesis rather than changes in the leucine content of proteins. The results demonstrate that the incorporation rate of [3H]leucine into a hot trichloroacetic acid-insoluble cell fraction can serve as an index of protein synthesis by bacterial assemblages in aquatic systems.     

Effect of diabetes on the rates of synthesis and degradation of ribosomes in rat muscle and liver in vivo

An important component of the decrease in protein synthesis in muscle of diabetic animals is a fall in the ribosome content. Therefore, we have investigated the turnover of ribosomes in skeletal muscle, heart, and liver of rats during the onset of diabetes. Synthesis rates were measured by incorporation of label into the protein moieties of the ribosomes, and a dual isotope technique was used to relate ribosome synthesis to that of total tissue protein. Degradation rates were calculated as the difference between the rates of synthesis and accumulation. The loss of ribosomes from gastrocnemius muscle and heart took place mainly between the 2nd and 4th days of insulin deficiency and was brought about largely by a very pronounced increase in the degradation rate, though synthesis also fell by a substantial amount. Rates of total tissue protein synthesis decreased markedly, but the degradation rates were only slightly elevated, if at all. Thus, the effect of diabetes on muscle ribosome breakdown was quite distinct from that on degradation of total tissue protein. In liver the response of protein synthesis to diabetes was much less pronounced than in muscle, and ribosome synthesis was not affected.     

Culture of honeybee organs: Development of a new medium and the importance of tracheation

Summary A culture system for honeybeen fat body and ovary was developed that supported optimal levels of protein synthesis by the explanted tissues. Abdominal body wall preparations of honeybee workers and queens, with adhering fat body, and ovaries of egg-laying queens were incubated in a culture medium designed to match honeybee hemolymph composition as closely as possible. Incorporation of [³H]eucine into soluble tissue proteins was measured. The new medium makes possible rates of tracer incorporation into fat body proteins that are up to three times higher than other media tested. When the tracheal system of the organs was let intact and open to the air during incubation, protein synthesis increased 17-fold (fat body) or 15-fold (ovary) as compared to preparations without open tracheas. After explantation into the medium, labeled proteins were synthesized at a highly variable rate for 10 h, probably due to wound response, and at a constant rate for the next 60 h. In contrast, ovarian protein synthesis occurred at a constant rate for at least 20 h and showed no wound response. The rate of tracer incorporation into fat body proteins was 3.2 times greater in tissues from the queen. This culture system is therefore suitable for a variety of investigations in honeybeen development and reproduction. These studies were supported by grants from the Deutsche Forschungsgemeinschaft, a Senior Scientist Award from the Alexander v. Humboldt Foundation for H. H. Hagedorn, and a fellowship from the Deutscher Akademischer Austauschdienst for H. H. Kaatz.     

Interrelation of aerobic glycolysis and lipogenesis in isolated perfused liver of well-fed rats

The rates of glycolysis and lipogenesis in isolated perfused liver of well-fed rats were studied. When liver was allowed to synthesize [14C]glycogen prior to perfusion, no more than 9% of the degraded [14C]glycogen was recovered in lactate and 6% in lipid. Addition of glucose, fructose and sorbitol enhanced concomitantly the formation of lactate and pyruvate and the rate of release of triglyceride and free fatty acid. Glucose was less efficient than fructose or sorbitol. The incorporation of 14C from these 14C-labelled substrates into lactate, pyruvate and lipids confirmed their role as carbon sources. Incorporation of 14C into the glycerol moiety of neutral lipid exceeded that found in the fatty acids, suggesting that these substrates contributed largely to the esterification of fatty acids. The total rate of de novo fatty acid synthesis was correlated with the formation of lactate and pyruvate. It is concluded that increased rates of aerobic glycolysis are related to increased rates of lipogenesis.