Developmentally induced changes in rat lung malic enzyme activities.

To determine lung malic enzyme activity at varying stages of development, both cytosolic and mitochondrial enzyme activities were assayed in rat lungs at various stages from day 16 of fetal life to 2 months of postnatal life by measuring the production of 14CO2 from 14C-malate. Malic enzyme activities were significantly higher in the mitochondrial than in the cytosolic fractions at all ages studied. The mitochondrial malic enzyme activity was significantly higher in canalicular stage (days 19-20) stage of lung development when compared to the glandular stage (days 16-18). The mitochondrial fraction at day 19 exhibited biphasic kinetics: high affinity, Km = 0.45 mmol, Vmax = 10.04 nmol/mg protein/min; and low affinity, Km = 5.48 mmol, Vmax = 56.83 nmol/mg protein/min. The cytosolic malic enzyme activity of all fetal stages (saccular stage [days 16-18], canalicular stage [days 19-20], and glandular stage [days 21-22] were significantly higher when compared to postnatal levels (postnatal days 1-10, adult). In contrast to the mitochondrial fraction, at day 19, the cytosolic fraction showed a single Km of 0.23 mmol, Vmax = 12.32 nmol/mg protein/min. The increased mitochondrial malic enzyme activity during late gestation would suggest that, as we have previously demonstrated, anaplerotic substrates other than glucose, may provide a significant energy source in fetal lung. The increased cytosolic activity in the prenatal phases would suggest that the NADPH provided from malic enzyme is an important contributor to de novo fatty acid synthesis, leading to surfactant synthesis, critical to normal lung development in late gestation.

The role of glutamine and other alternate substrates as energy sources in the fetal rat lung type II cell.

Glucose has been thought to be the primary substrate for energy metabolism in the developing lung; however, alternate substrates are used for energy metabolism in other organs. To examine the role of alternate substrates in the lung, we measured rates of oxidation of glutamine, glucose, lactate, and 3-hydroxybutyrate in type II pneumocytes isolated from d 19 fetal rat lungs by measuring the production of 14CO2 from labeled substrates. Glutamine had a rate of 24.36 +/- 4.51 nmol 14CO2 produced/ h/mg of protein (mean +/- SEM), whereas lactate had a significantly higher rate, 40.29 +/- 4.42. 3-Hydroxybutyrate had a rate of 14.91 +/- 1.93. The rate of glucose oxidation was 2.13 +/- 0.36, significantly lower than that of glutamine. To examine the interactions of substrates normally found in the intracellular milieu, we measured the effect of unlabeled substrates as competitors on labeled substrate. This identifies multiple metabolic compartments of energy metabolism. Glucose, but not lactate, inhibited the oxidation of glutamine, suggesting a compartmentation of tricarboxylic acid cycle activity, rather than simple dilution by glucose. Glucose and lactate had reciprocal inhibition. Our data suggest at least two separate compartments in the type II cells for substrate oxidation, one for glutamine metabolism and a second for glucose metabolism. In summary, we have documented that glutamine and other alternate substrates are oxidized preferentially over glucose for energy metabolism in the d 19 fetal rat lung type II pneumocyte. In addition, we have delineated some of the compartmentation that occurs within the developing type II cell, which may determine how these substrates are used.

The role of glutamine as an energy source in the developing rat lung.

Because multiple substrates have been shown to play a role in the metabolic homeostasis of different tissues, a series of studies were initiated to examine the role of alternate substrates in the lung. In these studies, we measured rates of oxidation of glutamine, glucose, lactate and 3-hydroxybutyrate in fibroblasts isolated from d 19 fetal rat lungs by measuring the production of 14CO2 from labeled substrates and compared them with earlier studies of isolated Type II cells. The rate of glutamine oxidation was 16.04 nmol 14CO2 x mg protein(-1) x hr(-1) in the fibroblasts compared with 24.36 in Type II cells. Three-hydroxybutyrate had a rate of 10.75 in the fibroblasts and 14.9 in the Type II cells. Lactate oxidation in fibroblasts was similar to that of glutamine, with a rate of 18.49; however, in Type II cells the rate of lactate oxidation was significantly higher at 40.29. Glucose was oxidized at a rate significantly lower than the other three substrates. In the fibroblasts, that rate was 1.22 and in Type II cells it was 2.13. To examine the interactions of substrates normally found in the intracellular milieu, we measured the effect of unlabeled substrates as competitors on labeled substrate in the fibroblasts, similar to our studies with Type II cells that identified multiple metabolic compartments of energy metabolism in these cell populations. Glucose, but not lactate, inhibited the oxidation of glutamine, suggesting a compartmentation of tricarboxylic acid cycle activity rather than simple dilution by glucose. Glucose and lactate had reciprocal inhibition in the Type II cells. Our data suggest at least two separate compartments in developing lung cells for substrate oxidation: one for glutamine metabolism and a second for glucose metabolism. In summary, we have documented that glutamine and other alternate substrates are oxidized preferentially over glucose for energy metabolism in the d 19 fetal rat lung.

Degrees of cooperativity between triiodothyronine and hydrocortisone in their regulation of the expression of myelin basic protein and proteolipid protein during brain development.

Cultures of cells dissociated from embryonic mouse cerebra were used to demonstrate: (1) that the developmental expression of the mRNA of proteolipid protein is dependent on thyroid hormone; (2) that the expression of the mRNA of proteolipid protein is stimulated not only by triiodothyronine but also by hydrocortisone, which achieve their respective stimulations by an additive and uncompetitive mechanism; (3) the stimulation of the net accumulation of the mRNA of myelin basic protein by hydrocortisone and triiodothyronine is also cooperative, additive, and uncompetitive, and (4) the stimulation of the net accumulation of myelin basic protein, during development by hydrocortisone, is completely dependent on the presence of thyroid hormone. These results suggest that the regulation of the synthesis of myelin basic protein by hydrocortisone requires the presence of triiodothyronine at a posttranscriptional event, but not for transcription itself.

In vivo studies on stereospecificity of the monoglyceride kinase, lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase and CDP-diglyceride synthase of Streptococcus mutans BHT using the stereoisomers of the ether lipid, dodecylglycerol.

Streptococcus mutans BHT metabolizes radioactive 3-dodecyl-sn-glycerol (sn-3-DDG) almost exclusively to lysophosphatidic acid, phosphatidic acid and 1,3-diradyl-sn-glycerol, whereas the cells of this organism metabolize 1-dodecyl-sn-glycerol (sn-1-DDG) to all of the glycerol lipids of S. mutans BHT, with the largest amounts incorporated into phosphatidylglycerol and diradylglycerol (mostly the 1,2- but also the 1,3-isomer). (The common names of lipids, such as phosphatidic acid, are used in the broader sense to mean that the lipid may contain alkyl as well as acyl groups.) The addition of an equivalent amount of nonradioactive sn-3-DDG to radioactive sn-1-DDG causes more of the radioactivity to accumulate at phosphatidic acid. These results indicate that the monoglyceride kinase (EC 2.7.1.94), lysophosphatidic acid acyltransferase (EC 2.3.1.40) and the monoglyceride acyltransferase (EC 2.3.1.22) enzymatic reactions are not stereospecific, and that the CDP-diglyceride synthase (EC 2.7.7.41) and phosphatidic acid phosphatase (EC 3.1.3.4) metabolic steps are stereospecific in S. mutans BHT. The synthesis of phosphatidic acid and lysophosphatidic acid from sn-3-DDG provides a unique method for synthesizing these glycerol lipids with the uncommon stereochemical configuration in which the phosphate moiety is in the sn-1 position.

Studies on the antibacterial activity of dodecylglycerol. Its limited metabolism and inhibition of glycerolipid and lipoteichoic acid biosynthesis in Streptococcus mutans BHT.

Growth-inhibitory concentrations of racemic sn-1(3)-dodecylglycerol inhibit the incorporation of [14C] glycerol into lipids and lipoteichoic acid of Streptococcus mutans BHT and alter the per cent composition of the glycerolipids. Increases in phosphatidic acid and diphosphatidylglycerol (at the expense of phosphatidylglycerol) contribute the most to the change in lipid composition. No cellular lysis occurs under these conditions. Radioactive racemic sn-1(3)-dodecylglycerol is readily taken up by the cell and is metabolized primarily to lysophosphatidic acid and phosphatidic acid with smaller amounts converted to phosphatidylglycerol and diacylglycerol. The accumulation of phosphatidic acid and the loss of viability respond in parallel to different concentrations of dodecylglycerol. An increase in CTP is also observed which together with the increase in phosphatidic acid suggests a possible impairment in the synthesis of CDP-diacylglycerol.

Mode of elongation of the glycerol phosphate polymer of membrane lipoteichoic acid of Streptococcus faecium ATCC 9790.

Specific degradation of membrane lipoteichoic acid of Streptococcus faecium ATCC 9790 by a phosphodiesterase from Aspergillus niger and by periodate oxidation has demonstrated that the enzymatic synthesis of the glycerol phosphate polymer of the molecule occurs by an external elongation system. Evidence of this type of mechanism was obtained with lipoteichoic acid synthesized in vivo or in vitro by differential radioisotope labeling techniques. The glycerol phosphate repeating units were transferred from phosphatidylglycerol and became linked through a phosphodiester bond to the glycerol phosphate unit of the chain farthest from or most external to the lipid end of the polymer.

Excretion of extracellular lipids by Streptococcus mutans BHT and FA-1.

Streptococcus mutans BHT and FA-1, when grown to log phase on chemically defined medium containing [14C]glycerol, excreted 15% of the total biosynthesized 14C-lipid into the medium. When grown to early stationary phase, 28 to 33% of the 14C-lipid was found in the medium. The radioactive lipids of these varieties of S. mutans were identified as diacylglycerol, diglucosyl diacylglycerol (DGD), monoglucosyl diacylglycerol, diphosphatidylglycerol, phosphatidylglycerol (PG), and smaller amounts of two other lipids tentatively were identified as amino acyl-PG and glycerol phosphoryl-DGD. All lipids were found as extracellular and intracellular components from cells grown to either log or stationary phase. However, there were some shifts in the relative percentage of these lipids as the cells changed from log to stationary phase. For example, the intracellular lipid content of log-phase S. mutans BHT was composed of 49% PG and 19% DGD, but these percents shifted to 18% PG and 57% DGD when the cells were grown to stationary phase. However, the extracellular lipids of this organism contained 50 to 60% PG and 20% DGD in both log and stationary phases.