Comparative utilization in vivo of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose and [1-C14] palmitate in the rat

Female rats were injected i.v. with comparable trace amounts of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose, or [1-14C] palmitate, and killed 30 min afterwards. The radioactivity remaining in plasma at that time was maximal in animals receiving [U-14C] glucose while the appearance of radioactive lipids was higher in the [U-14C] glycerol animals than in other groups receiving hydrosoluble substrates. The carcass, more than the liver, was the tissue where the greatest proportion of radioactivity was recovered, while the greatest percentage of radioactivity appeared in the liver in the form of lipids. The values of total radioactivity found in different tissues were very similar when using either labelled glucose or glycerol but the amount recovered as lipids was much greater in the latter. The maximal proportion of radioactive lipids appeared in the fatty-acid form in the liver, carcass, and lumbar fat pads when using [U-14C] glycerol as a hydrosoluble substrate, and the highest lipidic fraction appeared in adipose tissue as labelled, esterified fatty acids. In the spleen, heart, and kidney, most of the lipidic radioactivity from any of the hydrosoluble substrates appeared as glyceride glycerol. The highest proportion of radioactivity from [1-14C] palmitate appeared in the esterified fatty acid in adipose tissue, being followed in decreasing proportion by the heart, carcass, liver, kidney, and spleen. Thus at least in part, both labelled glucose and glycerol are used throughout different routes for their conversion in vivo to lipids. A certain proportion of glycerol is directly utilized by adipose tissue. The fatty acids esterification ability differs among the tissues and does not correspond directly with the reported activities of glycerokinase, suggesting that the alpha-glycerophosphate for esterification comes mainly from glucose and not from glycerol.     

[14C]acetylcholine synthesis and [14C]carbon dioxide production from [U-14C]glucose by tissue prisms from human neocortex.

1. [14C]Acetylcholine synthesis and 14CO2 production from [U-14C]glucose has been measured in tissue prism preparations from human neocortex. 2. Electron micrographs of prisms from human and rat neocortex show that both contain intact synaptic endings with evenly-distributed vesicles and normal-appearing mitochondria, but only poorly preserved cell body structure. 3. Synthesis of [14C]acetylcholine in prisms from rat neocortex is similar to estimates for turnover in vivo. Synthesis in prisms from human neocortex is 18% of that in rat tissue and 64% of that in tissue from baboon neocortex for incubations performed in 31 mM-K+. 4. Investigations of prisms prepared from rat brains stored at 37 degrees C after death revealed that synthesis of [14C]acetylcholine in the presence of 31 mM-K+ was greatly decreased within 30 min of post-mortem incubation, whereas synthesis at 5 mM-K+ and production of 14CO2 at both K+ concentrations were only significantly affected after longer periods. Changes were similar in neocortex and striatum. Thus human autopsy material is unlikely to be suitable for use with this system. 5. Investigations using animal models suggest that [14C]acetylcholine synthesis and 14CO2 production are not affected by surgical or anaesthetic procedures. 6. Neither [14C]acetylcholine synthesis nor 14CO2 production in human prisms was significantly changed with age between 15 and 68 years. 7. Samples from patients with the dementing condition Alzheimer's disease showed a significant decrease in [14C]acetylcholine synthesis to 47% of normal samples and a significant increase of 39% in production of 14CO2.     

Long-term metabolism of [U-14C]glucose in the whole rat brain

The experiment was performed on rats to which a single injection of [U-14C]glucose had been administered. Results were observed from the 7th to the 281st day following contamination. At 280 days only the lipids in the brain contained radioactivity, the highest degree of specific activity being found in the cerebrosides.     

Effect of D-galactosamine in vitro on [U-14C]palmitate oxidation, triacylglycerol synthesis and secretion in isolated hepatocytes

Isolated rat hepatocytes were used to study in vitro effects of 10 mM D-galactosamine (GalN) on hepatic fatty acids metabolism. At this concentration, membrane integrity and biochemical competence (i.e., gluconeogenesis and ureogenesis) remained unaffected. Protein synthesis and secretion, as measured by the incorporation of [U-14C]leucine into total and medium protein, was significantly inhibited when incubated for more than 2 h. GalN activated the incorporation of [U-14C]palmitate into triacylglycerols and depressed its utilization in the formation of labelled ketone bodies and 14CO2. Hepatocytes isolated from fasted rats exposed to GalN in vitro did not show any variation in prelabelled triacylglycerol secretion. GalN induced a rapid inhibition of prelabelled triacylglycerol secretion by hepatocytes isolated from fed rats in which this secretion occurred to a larger extent than in hepatocytes isolated from fasted rats. The data reported here suggest that GalN induces a rise of triacylglycerol synthesis by inhibiting the palmitate oxidation pathway and a decrease of triacylglycerol secretion through an early derangement of the secretory pathway.     

Enzymatic synthesis of [U-14C] ribose-labeled inosine.

A very potent anticholinesterase compound, 7-(diethoxyphosphinyloxy)-N-methylquinolinium fluorosulfate, has been used to determine the normality of acetylcholinesterase solutions. The inhibitor reacts rapidly and completely with acetylcholinesterase. The bimolecular rate constant is 2.5 × 10⁸m^?1 min^?1 and the equilibrium constant is about 10⁶. The reaction produces an inactive diethylphosphoryl enzyme in which the active serine is phosphorylated. The reaction produces the highly fluorescent 1-methyl-7-hydroxyquinolinium dipolar ion as a leaving group. The inhibited enzyme is quite stable and hydrolyzes to produce active enzyme only at the rate of 0.04%/min. The inhibitor was used in two ways for measuring the normality of acetylcholinesterase solutions: (1) The very fast reaction of the inhibitor with cholinesterase makes it convenient to determine the normality of enzyme solutions by measuring the decrease in enzyme activity caused by the addition of an accurately known quantity of the inhibitor. (2) The highly fluorescent nature of the leaving group makes it possible to measure the low concentration that is produced by the reaction of excess inhibitor with the enzyme. The two methods yielded activities per site of 6.9 × 10⁵ min^?1 and 7.3 × 10⁵ min^?1 using enzyme normalities of 1–2 × 10^?8m and 1–5 × 10^?m, respectively, using a commercial 11 S enzyme preparation from electric eel and acetylthiocholine as the enzyme substrate.     

Quantitative measurement of the L-type pentose phosphate cycle with [2-14C]glucose and [5-14C]glucose in isolated hepatocytes.

1. Investigations of the mechanism of the non-oxidative segment of the pentose phosphate cycle in isolatd hepatocytes by prediction-labelling studies following the metabolism of [2-14C]-, [5-14C]- and [4,5,6-14C]glucose are reported. The 14C distribution patterns in glucose 6-phosphate show that the reactions of the L-type pentose pathway in hepatocytes. 2. Estimates of the quantitative contribution of the L-type pentose cycle are the exclusive form of the pentose cycle to glucose metabolism have been made. The contribution of the L-type pentose cycle to the metabolism of glucose lies between 22 and 30% in isolated hepatocytes. 3. The distribution of 14C in the carbon atoms of glucose 6-phosphate following the metabolism of [4,5,6-14C]- and [2-14C]glucose indicate that gluconeogenesis from triose phosphate and non-oxidative formation of pentose 5-phosphate do not contribute significantly to randomization of 14C in isolated hepatocytes. The transaldolase exchange reaction between fructose 6-phosphate and glyceraldehyde 3-phosphate is very active in these cells.     

Measurement of exogenous carbohydrate oxidation: a comparison of [U-14C]glucose and [U-13C]glucose tracers

The purpose of this study was to assess the level of agreement between two techniques commonly used to measure exogenous carbohydrate oxidation (CHO(EXO)). To accomplish this, seven healthy male subjects (24 +/- 3 yr, 74.8 +/- 2.1 kg, V(O2(max)) 62 +/- 4 ml x kg(-1) x min(-1)) exercised at 50% of their peak power for 120 min on two occasions. During these exercise bouts, subjects ingested a solution containing either 144 g glucose (8.7% wt/vol glucose) or water. The glucose solution contained trace amounts of both [U-13C]glucose and [U-14C]glucose to allow CHO(EXO) to be quantified simultaneously. The water trial was used to correct for background 13C enrichment. 13C appearance in the expired air was measured using isotope ratio mass spectrometry, whereas 14C appearance was quantified by trapping expired CO(2) in solution (using hyamine hydroxide) and adding a scintillator before counting radioactivity. CHO(EXO) measured with [13C]glucose ([13C]CHO(EXO)) was significantly greater than CHO(EXO) measured with [14C]glucose ([14C]CHO(EXO)) from 30 to 120 min. There was a 15 +/- 4% difference between [13C]CHO(EXO) and [14C]CHO(EXO) such that the absolute difference increased with the magnitude of CHO(EXO). Further investigations suggest that the difference is not because of losses of CO2 from the trapping solution before counting or an underestimation of the "strength" of the trapping solution. Previous research suggests that the degree of isotopic fractionation is small (S. C. Kalhan, S. M. Savin, and P. A. Adam. J Lab Clin Med89: 285-294, 1977). Therefore, the explanation for the discrepancy in calculated CHO(EXO) remains to be fully understood.     

Differential labeling of glycerol moieties of phospholipids and triacylglycerols of cultured mammalian cells by [U-14 C] glucose.

Rabbit liver cells, in which fatty acid synthesis was suppressed by the rabbit serum component of the medium, were grown through 8- to 120-fold increases in cell numbers and mass of cell lipid in the presence of [U-14 C]-glucose. Triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine were isolated from the total cell lipid and deacylated. Carbons 1 and 3 of the glycerol from the triacylglycerols and the no. 1 glycerol carbons of the two deacylated phospholids were oxidized by periodate and isolated as the dimedon derivative of formaldehyde. The specific activities of the glycerol carbons indicated that 58, 44, and 37 percent of the glycerol of the triacylglycerols. phosphatidylcholine, and phosphatidylethanolamine, respectively, were derived from the glucose of the medium. An additional 8 percent and 1-2 percent of the glycerol of each lipid was derived, respectively, from [U-14 C] glycerol and U14C-labeled amino acids added to the medium. In agreement with an experiment with albumin-bound [9,10- minus 3H]-oleic acid, and with smilar earlier experiments, it appears likely that appriacylglycerols originated from serum lipoproteins, or their partial hydrolysis products. An appreciable part of the ethanolamine of the cells' phosphatidylethanolamine originated from exogenous U- minus 14 C-labeled amino acids. Phosphatidyl-ethanolamine, however, was not a primary source of phosphatidylcholine. Labeling of the fatty acids of triacylglycerols and phospholipids by radioactive glucose, glycerol and amino acids was negligible.     

Anomeric specificity of D-[U-14C]glucose incorporation into glycogen in rat hemidiaphragms

The anomeric specificity of D-[U-14C]glucose incorporation into glycogen in rat hemidiaphragms was investigated. For this purpose, the hemidiaphragms were preincubated for 30 min at 37 degrees C and then incubated for 5 min at the same temperature in the presence of alpha- or beta-D-[U-14C]glucose. The concentrations of D-glucose (5.6 or 8.8 mM) and insulin (0 or 10 mU/ml) were identical during the preincubation and incubation periods. The incubation medium was prepared in D2O/H2O (3:1, v/v) in order to delay the interconversion of the D-glucose anomers. In addition to glycogen labelling, the output of radioactive acidic metabolites was also measured. Insulin caused a preferential stimulation of glycogen labelling relative to glycolysis. Such was not the case in response to a rise in D-glucose concentration. At 5.6 mM D-glucose and whether in the presence or absence of insulin, both glycogen labelling and glycolysis were lower with alpha-D-glucose than with beta-D-glucose suggesting a higher rate of beta-D-glucose than alpha-D-glucose transport across the plasma membrane. A mirror image was found at 8.8 mM D-glucose, especially in the absence of insulin. At this close-to-physiological hexose concentration, insulin lowered the alpha/beta ratio for glycogen labelling. On the contrary, the rise in D-glucose concentration increased such a ratio. Since such a rise is probably little affected by any possible anomeric difference in D-glucose transport across the plasma membrane, the present results strongly suggest that the intracellular factors regulating net glycogen synthesis, as well as glycolytic flux, display obvious preference for alpha-D-glucose.     

Fate of [G-3H]glutamate and [U-14C]glucose in the rat liver in vivo

• 1.1. After injection of a mixture of [G-³H]glutamate and [U-¹⁴C]glucose to rats, the highest amount of ¹⁴C was found in an unidentified compound (glycopeptide?) of the acid soluble extract of the liver at 2 min.
• 2.2. With increasing time after the injection the specific radioactivity of [³H]glutamate decreased and that of [³H]glutamine increased in the liver.
• 3.3. The labelling of the liver protein with ¹⁴C was due to [¹⁴C]glutamate and [¹⁴C]aspartate, and that with ³H was exclusively due to [³H]glutamate.

     

Convenient preparative synthesis of [14C]trehalose from [14C]glucose by intact Escherichia coli cells.

At high osmolarity, Escherichia coli synthesizes trehalose intracellularly, irrespective of the nature of the carbon source. Synthesis proceeds via the transfer of UDP-glucose to glucose 6-phosphate, yielding trehalose 6-phosphate, followed by its dephosphorylation to trehalose (H.M. Giaeyer, B.O. Styrvold, I. Kaasen, and A.R. Strøm, J. Bacteriol. 170:2841-2849, 1988). This reaction was exploited to preparatively synthesize [14C]trehalose from exogenous [14C]glucose by using intact bacteria of a mutant (DF214) that could not metabolize glucose. The total yield of radiochemically pure trehalose from glucose was routinely more than 50%.     

Measurement of mouse brain glucose utilization in vivo using [U-14C]glucose

The rate of [2-¹⁴C]glucose uptake has been used as an indication of the status of energy consumption by the rat brain, but the cost of this radiolabel can be prohibitive and the surgical manipulation involved in published methods is extensive. A method for measuring glucose utilization in vivo in mouse brain with [U-¹⁴C]glucose is described in this article. Glucose consumption in whole mouse brain obtained with [U-¹⁴C]glucose or [2-¹⁴C]glucose was 0.650±0.022 and 0.716±0.36 nmol/mg/min, respectively. In all instances the rate obtained with the uniformly labeled isotope was somewhat lower than that found with [2-¹⁴C]glucose. The rate of glucose utilization measured with either isotope was significantly depressed in sodium pentobarbital anesthetized mice. The method described here is advantageous because [U-¹⁴C]glucose is substantially less expensive than [2-¹⁴C]glucose and surgical intervention is avoided.     

The biosynthesis of phosphatidylserine and phosphatidylethanolamine from L-[3-14C]serine in isolated rat hepatocytes

Incorporation of L-[3-14C]serine into phosphatidylserine (PS) and phosphatidylethanolamine (PE) has been studied in isolated rat hepatocytes. Ethanolamine inhibited the incorporation, indicating competition with serine in the base-exchange reaction. Choline, monomethylethanolamine, dimethylethanolamine and dimethyl-3-aminopropan-1-ol had no such effect. The observed rate of PS biosynthesis corresponded to 7-17 nmol/min per liver at 0.55 mM L-serine. The results indicate that only a small fraction (1/25 to 1/70) of the PS pool equilibrates with the base-exchange enzyme, and that decarboxylation to PE occurs preferentially from this pool. The rate of PS synthesis and decarboxylation can therefore not be calculated by methods which assume random, homogeneous labelling of the total PS pool. The apparent rate of PS decarboxylation increased approx. 4-fold when L-serine increased from 0.5 to 2.25 mM, suggesting that decarboxylation of PS to PE might be regulated by the concentration of L-serine or by the amount of PS present in the hepatocyte cell membranes. Lauric, palmitic, stearic, oleic and linoleic acid decreased the rate of PS synthesis. At 0.5 mM, lauric and palmitic acid were most inhibitory. At 1.0 mM, linoleic acid was the least inhibitory fatty acid. The saturated hexaenoic and saturated tetraenoic species of PS contained 51 and 29%, respectively, of the incorporated L-[3-14C]serine. The combined monoene dienoic/diene dienoic fraction had the highest rate of synthesis judged by its relative specific activity. At 0.9 mM concentration, linoleic acid doubled the relative specific activity of the combined monoene dienoic/diene dienoic fraction of PS. Incorporation of L-[3-14C]serine into molecular species of PE resembled that into PS, both in the absence and presence of linoleic acid, suggesting that the phosphatidylserine decarboxylase (EC 4.1.1.65) has a low specificity towards the fatty acid composition of PS. The results indicate that biosynthesis of PS from L-serine occurs mainly by the base-exchange with only negligible contribution from direct incorporation of phosphatidic acid or diacylglycerol. Furthermore, the deacylation-reacylation pathway seem to contribute only little to the determination of the fatty acid composition of hepatocyte PS. Active PS turnover seems to be confined to a small fraction of the PS pool.     

Convenient preparative synthesis of [14C]trehalose from [14C]glucose by intact Escherichia coli cells

At high osmolarity, Escherichia coli synthesizes trehalose intracellularly, irrespective of the nature of the carbon source. Synthesis proceeds via the transfer of UDP-glucose to glucose 6-phosphate, yielding trehalose 6-phosphate, followed by its dephosphorylation to trehalose (H.M. Giaeyer, B.O. Styrvold, I. Kaasen, and A.R. Str?m, J. Bacteriol. 170:2841-2849, 1988). This reaction was exploited to preparatively synthesize [14C]trehalose from exogenous [14C]glucose by using intact bacteria of a mutant (DF214) that could not metabolize glucose. The total yield of radiochemically pure trehalose from glucose was routinely more than 50%.     

Intermediates of peroxisomal beta-oxidation of [U-14C]hexadecanedionoate. A study of the acyl-CoA esters which accumulate during peroxisomal beta-oxidation of [U-14C]hexadecanedionate and [U-14C]hexadecanedionoyl-mono-CoA.

1. The oxidation of [U-14C]hexadecanedionoyl-mono-CoA was stimulated by CoA, by carnitine in the absence of CoA and by the presence of an NAD(+)-regenerating system. 2. Substrate inhibition was observed with respect to [U-14C]hexadecanedionoyl-mono-CoA at concentrations greater than 35 microM. 3. Acetyl-CoA and the dicarboxyl-CoA esters of chain length C6-16 were detected by HPLC under standard incubation conditions. 4. In the absence of the NAD(+)-regenerating system, 2-enoyl-CoA and 3-hydroxacyl-CoA esters were detected. 5. In general, the peroxisomal beta-oxidation of dicarboxylates is very similar to that of monocarboxylates [Bartlett, K., Hovik, R., Eaton, S., Watmough, N. J. & Osmundsen, H. (1990) Biochem. J. 270, 175-180] except that chain shortening does not proceed beyond C6. 6. We conclude that the peroxisomal beta-oxidation of dicarboxylates is regulated by the redox state of the peroxisomal matrix and CoA availability.     

Hormonal induction of alpha2u-globulin synthesis in isolated rat hepatocytes.

Hepatocytes freshly prepared with collagenase synthesize alpha 2u-globulin and other hepatic proteins in vitro at approximately the same rate throughout 30 h of incubation. The newly synthesized proteins are efficiently secreted into the medium throughout this period. That the secretion of proteins by hepatocytes is not due to cell leakage is shown by the fact that 30 micrometer colchicine prevents the appearance of labeled alpha2u-globulin and other proteins in the medium. Hepatocytes isolated from animals in different endocrine states synthesize alpha2u-globulin in vitro at rates consistent with the hormonal effects upon its in vivo biosynthesis. In vitro addition of androgens to hepatocytes isolated from castrated male rats evokes an elevation of general protein synthesis in these hepatocytes. Glucocorticoids, added in vitro, specifically induce and elevated rate of alpha2u-globulin synthesis.