Network flux analysis: impact of 13C-substrates on metabolism in Arabidopsis thaliana cell suspension cultures

The aim of this study was to test the assumption that (13)C-enrichment of respiratory substrate does not perturb metabolism. Cell suspension cultures of Arabidopsis thaliana were grown in MS medium containing unlabelled glucose (with (13)C at natural abundance), 100% [1-(13)C]glucose, 100% [U-(13)C(6)]glucose or 10% [U-(13)C(6)]glucose plus 90% unlabelled glucose. There was no significant difference in the metabolism of [U-(14)C]glucose between the cultures. Similarly, the pattern of (14)CO(2) release from specifically labelled [(14)C]-substrates was unaffected. Principal component analysis of (13)C-decoupled (1)H NMR metabolite fingerprints of cell extracts was unable to discriminate between the different culture conditions. It is concluded that (13)C-enrichment of the growth substrate has no effect on flux through the central pathways of carbon metabolism in higher plants. This conclusion supports the implicit assumption in metabolic flux analysis that steady-state (13)C-labelling does not perturb fluxes through the reactions of the metabolic network it seeks to quantify.     

Radiorespirometry evidence for the discrimination between 13C-enriched glucose and unlabelled glucose molecules by Paracoccus denitrificans

Paracoccus denitrificans was grown on either unlabelled glucose, [1-13C]glucose or [6-13C]glucose as the sole carbon source for growth. The cells were then incubated with a range of 14C-glucose substrates to compare the 14CO2-evolution rates between cells grown on the glucose and the 13C-labelled glucose. Cells grown on 13C-glucose had significantly faster rates of 14CO2-evolution than those grown on unlabelled glucose. The % yields of 14CO2, per [1-14C]-, [6-14C]- and [U-14C]glucose supplied were also substantially greater than those measured for cells grown on unlabelled glucose. The data indicated that growth of Paracoccus on 13C-enriched glucose substrates resulted in cells with notably different 14C-glucose oxidation metabolism compared to that observed in cells grown on unlabelled glucose.     

The fate of isotopic carbon in kidney cortex synthesizing glucose from lactate

1. When slices of kidney cortex are incubated with lactate and acetoacetate, lactate is almost quantitatively converted into glucose whereas acetoacetate provides a major part of the fuel of respiration. 2. In apparent contrast with these findings a large fraction of ¹⁴C-labelled acetoacetate appears in glucose and a large fraction of ¹⁴C-labelled lactate appears in the carbon dioxide. 3. The findings can be explained by: (a) the participation of oxaloacetate as an intermediate of both gluconeogenesis and respiration; (b) the fact that the carbon dioxide formed in the course of one turn of the tricarboxylic acid cycle is exclusively derived from oxaloacetate. 4. As a result there is a `crossing over' of the carbon of the substrate of respiration to the pathway of gluconeogenesis and of the carbon of the glucogenic precursors to the pathway of respiration. 5. In the given situation the fate of the label does not allow predictions to be made about the net fate of the labelled metabolites. 6. The implications of the findings on the interpretation of isotopic data are discussed.     

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.     

Rate studies of polysaccharide biosynthesis from guanosine diphosphate α-d-glucose and guanosine diphosphate α-d-mannose

With enzyme preparations from Phaseolus aureus seedlings, the initial rate of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]glucose is not increased by additions of GDP-alpha-d-mannose. However, final incorporation is increased by addition of GDP-alpha-d-mannose, since the total reaction-time is extended. In contrast, the initial rate of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]mannose is increased by all concentrations of GDP-alpha-d-glucose that are less than that of the GDP-alpha-d-[(14)C]mannose. Maximum stimulation of the initial rate occurs at a GDP-alpha-d-[(14)C]mannose/GDP-alpha-d-glucose concentration ratio of about 4:1. However, eventual incorporation from GDP-alpha-d-[(14)C]mannose is decreased by the addition of GDP-alpha-d-glucose, since the reaction rate falls off sharply after about 2min. Reciprocal plots of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]mannose result in biphasic graphs. The two straight-line portions of the plot are joined by a curved line in the concentration range between 2-3 and 50mum. Extrapolated K(m) values for the two linear components are 0.4-1.0 and 700-1500mum. The effect of GDP-alpha-d-glucose on the kinetics of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]mannose is complex, and depends on relative concentrations of the two sugar nucleotides. (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]glucose also results in biphasic reciprocal plots. One component appears to have K(m) about 2-3mum, the other about 200-400mum. In this reaction, GDP-alpha-d-mannose appears to be a competitive inhibitor with K(i) 20-30mum. With particulate preparations of P. aureus, GDP-alpha-d-[(14)C]glucose appears to be a precursor for the synthesis of one polysaccharide, a glucomannan, the mannose moieties of which are derived from an intermediate existing in the particulate preparation. From the rate results, GDP-alpha-d-[(14)C]mannose appears to be a precursor for at least two polysaccharides, one of which is a glucomannan.     

Loss of [13C]glycerol carbon via the pentose cycle. Implications for gluconeogenesis measurement by mass isotoper distribution analysis

Whereas many reports substantiated the suitability of using [2-(13)C]glycerol and Mass Isotoper Distribution Analysis for gluconeogenesis, the use of [(13)C]glycerol had been shown to give lower estimates of gluconeogenesis (GNG). The reason for the underestimation has been attributed to asymmetric isotope incorporation during gluconeogenesis as well as zonation of gluconeogenic enzymes and a [(13)C]glycerol gradient across the liver. Since the cycling of glycerol carbons through the pentose cycle pathways can introduce asymmetry in glucose labeling pattern and tracer dilution, we present here a study of the role of the pentose cycle in gluconeogenesis in Fao cells. The metabolic regulation of glucose release and gluconeogenesis by insulin was also studied. Serum-starved cells were incubated for 24 h in Dulbecco's modified Eagle's media containing 1.5 mm [U-(13)C]glycerol. Mass isotopomers of whole glucose from medium or glycogen and those of the C-1-C-4 fragment were highly asymmetrical, typical of that resulting from the cycling of glucose carbon through the pentose cycle. Substantial exchange of tracer between hexose and pentose intermediates was observed. Our results offer an alternative mechanism for the asymmetrical labeling of glucose carbon from triose phosphate. The scrambling of (13)C in hexose phosphate via the pentose phosphate cycle prior to glucose release into the medium is indistinguishable from dilution of labeled glucose by glycogen using MIDA and probably accounts for the underestimation of GNG using (13)C tracer methods.     

The metabolism of glucose 6-phosphate by mammalian cerebral cortex in vitro

1. The metabolism of glucose 6-phosphate in rat cerebral-cortex slices in vitro was compared with that of glucose. It was found that a glucose 6-phosphate concentration of 25mm was required to achieve maximal oxygen uptake rates and ATP concentrations, whereas only 2mm-glucose was required. 2. When 25mm-[U-(14)C]glucose 6-phosphate was used as substrate, the pattern of labelling of metabolites was found to be quantitatively and qualitatively similar to the pattern found with 10mm-[U-(14)C]glucose, except that incorporation into [(14)C]lactate was decreased, and significant amounts of [(14)C]glucose and [(14)C]mannose phosphate and [(14)C]fructose phosphate were formed. 3. Unlabelled glucose (10mm) caused a tenfold decrease in the incorporation of 25mm-[U-(14)C]glucose 6-phosphate into all metabolites except [(14)C]glucose and [(14)C]mannose phosphate and [(14)C]fructose phosphate. In contrast, unlabelled glucose 6-phosphate (25mm) had no effect on the metabolism of 10mm-[U-(14)C]glucose other than to increase markedly the incorporation into, and amount of, [(14)C]lactate, the specific radioactivity of this compound remaining approximately the same. 4. The effect of glucose 6-phosphate in increasing lactate formation from glucose was found to occur also with a number of other phosphate esters and with inorganic phosphate. Further investigation indicated that the effect was probably due to binding of medium calcium by the phosphate moiety, thereby de-inhibiting glucose uptake. 5. Incubations carried out in a high-phosphate high-potassium medium gave a pattern of metabolism similar to that found when slices were subjected to depolarizing conditions. Tris-buffered medium gave similar results to bicarbonate-buffered saline, except that it allowed much less lactate formation from glucose. 6. Part of the glucose formed from glucose 6-phosphate was extracellular and was produced at a rate of 12mumol/h per g of tissue in Krebs tris medium when glycolysis was blocked. The amount formed was much less when 25mm-P(i) or 26mm-HCO(3) (-) was present, the latter being in the absence of tris. 7. Glucose 6-phosphate also gave rise to an intracellular glucose pool, whereas no intracellular glucose was detectable when glucose was the substrate.     

Demethylation of [14C]-labelled veratric acid and oxidation of methanol and formaldehyde by the white-rot fungus Phlebia radiata

Veratric acids 14C-labelled in carboxyl group, 3-OCH3, 4-OCH3, or aromatic ring together with unlabelled veratric acid were supplemented in the cultures of the white-rot fungus Phlebia radiata. The effect of various carbon sources on the release of 14CO2 was studied. Veratric acid was readily decarboxylated, maximally already on day 1 from the addition of [14COOH]-veratric acid. High amounts (4%) of glucose slightly repressed the decarboxylation. In medium supplemented with cellulose the methoxyl group in position 4 was much more readily mineralized to CO2 than the group in position 3. The maximum evolution was achieved on day 5, two days from the addition. Cellulose did not repress methanol oxidation but repression of methanol oxidation by glucose was detected in media supplemented with [O14CH3]-veratric acids and 14CH3OH. However, glucose did not repress oxidation of H14CHO. The apparent uptake of 14C by fungal mycelium, especially from methoxyl groups, but also from the aromatic ring, may partially be due to the strong slime formation observed in cellobiose medium. Also in cellobiose medium apparent uptake of 14C from 14C-labelled methoxyl groups was observed.     

The influence of electrical stimulation in vitro on protein synthesis and other metabolic parameters of rat extensor digitorum longus muscle

1. Apparatus is described in which rat extensor digitorum longus muscle can be incubated in buffer under conditions of light tension and be subject to contractures induced by electrical stimulation in vitro. Under these conditions the tissue retains its weight, its content of potassium and size of the extracellular space at values similar to those in vivo. 2. Though uptake of glucose was enhanced on addition of insulin, there was little increase in glucose consumption on stimulation. Breakdown of glycogen and enhancement of lactate output were found on stimulation. 3. Incorporation into protein of several labelled amino acids was diminished during stimulation. Accumulation of [(14)C]leucine was enhanced whereas that of glycine was decreased. 4. There were no very consistent changes in the content of free unlabelled amino acids during incubation with or without stimulation. Comparison of actual amino acid concentrations in tissue and incubation mixture with accumulation of (14)C-labelled amino acid indicated that full equilibration of the cell pool of amino amino acids with the medium is slow. 5. Substantial oxidation of several (14)C-labelled acids was observed. 6. The ATP content of the tissue declined a little during incubation and somewhat faster after a period of stimulation. 7. The results are discussed in relation to the way in which exercise can induce muscle hypertrophy.     

Fatty acid biosynthesis by a particulate preparation from germinating pea

1. Fatty acid synthesis was studied in microsomal preparations from germinating pea (Pisum sativum). 2. The preparations synthesized a mixture of saturated fatty acids up to a chain length of C(24) from [(14)C]malonyl-CoA. 3. Whereas hexadecanoic acid was made de novo, octadecanoic acid and icosanoic acid were synthesized by elongation. 4. The products formed during [(14)C]malonyl-CoA incubation were analysed, and unesterified fatty acids and polar lipids were found to be major products. [(14)C]Palmitic acid represented a high percentage of the acyl-carrier protein esters, whereas (14)C-labelled very-long-chain fatty acids were mainly present as unesterified fatty acids. CoA esters were minor products. 5. The addition of exogenous lipids to the incubation system usually resulted in stimulation of [(14)C]malonyl-CoA incorporation into fatty acids. The greatest stimulation was obtained with dipalmitoyl phosphatidylcholine. Both exogenous palmitic acid and dipalmitoyl phosphatidylcholine increased the amount of [(14)C]-stearic acid synthesized, relative to [(14)C]palmitic acid. Addition of stearic acid increased the amount of [(14)C]icosanoic acid formed. 6. [(14)C]Stearic acid was elongated more effectively to icosanoic acid than [(14)C]stearoyl-CoA, and its conversion was not decreased by addition of unlabelled stearoyl-CoA. 7. Incorporation of [(14)C]malonyl-CoA into fatty acids was markedly decreased by iodoacetamide and 5,5'-dithiobis-(2-nitrobenzoic acid). Palmitate elongation was sensitive to arsenite addition, and stearate elongation to the presence of Triton X-100 or fluoride. The action of fluoride was not, apparently, due to chelation. 8. The microsomal preparations differed from soluble fractions from germinating pea in (a) synthesizing very-long-chain fatty acids, (b) not utilizing exogenous palmitate-acyl-carrier protein as a substrate for palmitate elongation and (c) having fatty acid synthesis stimulated by the addition of certain complex lipids.     

The pathway from arachidonic to docosapentaenoic acid (20:4n-6 to 22:5n-6) and from eicosapentaenoic to docosahexaenoic acid (20:5n-3 to 22:6n-3) studied in testicular cells from immature rats

The concentration-dependent metabolism of 1-(14)C-labelled precursors of 22:5n-6 and 22:6n-3 was compared in rat testis cells. The amounts of [(14)C]22- and 24-carbon metabolites were measured by HPLC. The conversion of [1-(14)C]20:5n-3 to [3-(14)C]22:6n-3 was more efficient than that of [1-(14)C]20:4n-6 to [3-(14)C]22:5n-6. At low substrate concentration (4 microM) it was 3.4 times more efficient, reduced to 2.3 times at high substrate concentration (40 microM). The conversion of [1-(14)C]22:5n-3 to [1-(14)C]22:6n-3 was 1.7 times more efficient than that of [1-(14)C]22:4n-6 to [1-(14)C]22:5n-6 using a low, but almost equally efficient using a high substrate concentration. When unlabelled 20:5n-3 was added to a cell suspension incubated with [1-(14)C]20:4n-6 or unlabelled 22:5n-3 to a cell suspension incubated with [1-(14)C]22:4n-6, the unlabelled n-3 fatty acids strongly inhibited the conversion of [1-(14)C]20:4n-6 or [1-(14)C]22:4n-6 to [(14)C]22:5n-6. In the reciprocal experiment, unlabelled 20:4n-6 and 22:4n-6 only weakly inhibited the conversion of [1-(14)C]20:5n-3 and [1-(14)C]22:5n-3 to [(14)C]22:6n-3. The results indicate that if both n-6 and n-3 fatty acids are present, the n-3 fatty acids are preferred over the n-6 fatty acids in the elongation from 20- to 22- and from 22- to 24-carbon atom fatty acids. In vivo the demand for 22-carbon fatty acids for spermatogenesis in the rat may exceed the supply of n-3 precursors and thus facilitate the formation of 22:5n-6 from the more abundant n-6 precursors.     

The regulation of triglyceride synthesis and fatty acid synthesis in rat epididymal adipose tissue. Effects of insulin, adrenaline and some metabolites in vitro

1. Adipose tissues from rats fed a balanced diet were incubated in the presence of glucose (20mm) with the following additions: insulin, anti-insulin serum, insulin+acetate, insulin+pyruvate, insulin+lactate, insulin+phenazine methosulphate, insulin+oleate+albumin, insulin+adrenaline+albumin, insulin+6-N-2'-O-dibutyryl 3':5'-cyclic AMP+albumin. 2. Measurements were made of the whole tissue concentrations of adenine nucleotides, hexose phosphates, triose phosphates, glycerol 1-phosphate, 3 phosphoglycerate, 6-phosphogluconate, long-chain fatty acyl-CoA, acid-soluble CoA, citrate, isocitrate, malate and 2-oxoglutarate, and of the release into the incubation medium of lactate, pyruvate and glycerol after 1h of incubation. 3. Fluxes of [(14)C]glucose carbon through the major pathways of glucose metabolism were calculated from the yields of (14)C in various products after 2h of incubation. Fluxes of [(14)C]acetate, [(14)C]pyruvate or [(14)C]lactate carbon in the presence of glucose were also determined. 4. Measurements were also made of the whole-tissue concentrations of metabolites in tissues taken directly from Nembutal-anaesthetized rats. 5. Whole tissue mass-action ratios for phosphofructokinase, phosphoglucose isomerase and the combined (aldolasextriose phosphate isomerase) reaction were similar in vivo and in vitro. The reactants of phosphofructokinase appeared to be far from mass-action equilibrium. In vitro, the reactants of hexokinase also appeared to be far from mass-action equilibrium. 6. Correlation of observed changes in glycolytic flux with changes in fructose 6-phosphate concentration suggested that phosphofructokinase may show regulatory behaviour. The enzyme appeared to be activated in the presence of oleate or adrenaline and to be inhibited in the presence of lactate or pyruvate. 7. Evidence is presented that the reactants of lactate dehydrogenase and glycerol 1-phosphate dehydrogenase may be near to mass-action equilibrium in the cytoplasm. 8. No satisfactory correlations could be drawn between the whole-tissue concentrations of long-chain fatty acyl-CoA, citrate and glycerol 1-phosphate and the observed rates of triglyceride and fatty acid synthesis. Under the conditions employed, the concentration of glycerol 1-phosphate appeared to depend mainly on the cytoplasmic [NAD(+)]/[NADH] ratios. 9. Calculated hexose monophosphate pathway flux rates roughly correlated with fatty acid synthesis rates and with whole tissue [6-phosphogluconate]/[glucose 6-phosphate] ratios. The relative rates of production of NADPH for fatty acid synthesis by the hexose monophosphate pathway and by the ;malic enzyme' are discussed. It is suggested that all NADH produced in the cytoplasm may be used in that compartment for reductive synthesis of fatty acids, lactate or glycerol 1-phosphate.     

The catabolism of phosphatidylethanolamine by the rumen protozoon Entodinium caudatum and its conversion into the N-(1-carboxyethyl) derivative

1. The N-(2-hydroxyethyl)alanine esterified to phosphatidic acid in anaerobic ciliate rumen protozoa has the l configuration. 2. Labelling experiments with Entodinium caudatum cultures using [(32)P]P(i) [2-(14)C]ethanolamine and (32)P- and (14)C-labelled phosphatidylethanolamine show that phosphatidylethanolamine is the direct lipid precursor of the N-(2-hydroxyethyl)alanine-containing phospholipid. 3. Labelling experiments with [(14)C]starch, [(14)C]lactate and [(14)C]pyruvate with E. caudatum cultures indicate that a three-carbon glycolytic intermediate is probably the precursor of the N-(1-carboxyethyl) grouping which substitutes on the amino group of phosphatidylethanolamine. 4. [(32)P]phosphatidylethanolamine is catabolized by E. caudatum forming initially glycerylphosphorylethanolamine and subsequently glycerophosphate and P(i). A little phosphorylethanolamine formed may possibly arise from bacterial enzymes ingested by the protozoa.     

Biosynthesis of amino acids in Clostridium pasteurianum

1. Clostridium pasteurianum was grown on a synthetic medium with the following carbon sources: (a) (14)C-labelled glucose, alone or with unlabelled aspartate or glutamate, or (b) unlabelled glucose plus (14)C-labelled aspartate, glutamate, threonine, serine or glycine. The incorporation of (14)C into the amino acids of the cell protein was examined. 2. In both series of experiments carbon from exogenous glutamate was incorporated into proline and arginine; carbon from aspartate was incorporated into glutamate, proline, arginine, lysine, methionine, threonine, isoleucine, glycine and serine. Incorporations from the other exogenous amino acids indicated the metabolic sequence: aspartate --> threonine --> glycine right harpoon over left harpoon serine. 3. The following activities were demonstrated in cell-free extracts of the organism: (a) the formation of aspartate by carboxylation of phosphoenolpyruvate or pyruvate, followed by transamination; (b) the individual reactions of the tricarboxylic acid route to 2-oxoglutarate from oxaloacetate; glutamate dehydrogenase was not detected; (c) the conversion of aspartate into threonine via homoserine; (d) the conversion of threonine into glycine by a constitutive threonine aldolase; (e) serine transaminase, phosphoserine transaminase, glycerate dehydrogenase and phosphoglycerate dehydrogenase. This last activity was abnormally high. 4. The combined evidence indicates that in C. pasteurianum the biosynthetic role of aspartate and glutamate is generally similar to that in aerobic and facultatively aerobic organisms, but that glycine is synthesized from glucose via aspartate and threonine.     

Sub-compartmentation of the 'cytosolic' glucose 6-phosphate pool in cultured rat hepatocytes

[14C]Glucose release either from endogenous 14C-prelabelled glycogen or from added 14C-labelled glucose 6-phosphate was measured in filipin-treated, permeabilized hepatocytes in 48 h culture. [14C]Glucose output from prelabelled glycogen was not altered by the addition of 5 mM glucose 6-phosphate to the incubation medium. Conversely, [14C]glucose release from 5 mM labelled glucose 6-phosphate was not influenced by different glycogen concentrations in the cells. Moreover, in the permeabilized cells the anion transport inhibitor DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) inhibited only the liberation of [14C]glucose from labelled glucose 6-phosphate but not from glycogen. It is therefore concluded that there exist at least 2 separate, mutually non-accessible glucose 6-phosphate pools in cultured rat hepatocytes, one linked to glycogenolysis and the other to gluconeogenesis.     

13C-NMR study of glycerol metabolism in rabbit renal cells of proximal convoluted tubules

Perchloric acid extracts of rabbit renal proximal convoluted tubular cells (PCT) incubated with [2-13C]glycerol and [1,3-13C]glycerol were investigated by 13C-NMR spectroscopy. These 13C-NMR spectra enabled us to determine cell metabolic pathways of glycerol in PCT cells. The main percentage of 13C-label, arising from 13C-enriched glycerol, was found in glucose, lactate, glutamine and glutamate. So far it can be concluded that glycerol is a suitable substrate for PCT cells and is involved in gluconeogenesis and glycolysis as well in the Krebs cycle intermediates. Label exchange and label enrichment in 13C-labelled glucose, arising from [2-13C]glycerol and [1,3-13C]glycerol, is explained by label scrambling through the pentose shunt and a label exchange in the triose phosphate pool. From relative enrichments it is estimated that the ratio of the pyruvate kinase flux to the gluconeogenetic flux is 0.97:1 and that the ratio of pyruvate carboxylase activity relative to pyruvate dehydrogenase activity is 2.0:1. Our results show that 13C-NMR spectroscopy, using 13C-labelled substrates, is a powerful tool for the examination of renal metabolism.     

Metabolism of the dimethyl ester of [2,3-13C]succinic acid in rat hepatocytes

Hepatocytes prepared from overnight fasted rats were incubated for 120 min in the presence of the dimethyl ester of [2,3-13C]succinic acid (10 mM). The identification and quantification of 13C-enriched metabolites in the incubation medium were performed by a novel computational strategy for the deconvolution of NMR spectra with multiplet structures and constraints. The generation of 13C-labelled metabolites, including succinate, fumarate, malate, lactate, alanine, aspartate and glucose accounted for about half of the initial amount of the ester present in the incubation medium. A fair correlation was observed between the experimental abundance of each 13C-labelled glucose isotopomer and the corresponding values derived from a model for the metabolism of [2,3-13C]succinate. Newly formed glucose was more efficiently labelled in the carbon C5 than C2, as well as the carbon C6 than C1, supporting the concept that D-glyceraldehyde-3-phosphate may undergo enzyme-to-enzyme channelling between glyceraldehyde-3-phosphate dehydrogenase and phosphofructoaldolase.     

Role of microtubules in the regulation of metabolism in isolated cerebral microvessels

We used¹³C-labeled substrates and nuclearmagnetic resonance spectroscopy to examine carbohydrate metabolism invascular smooth muscle of freshly isolated pig cerebral microvessels(PCMV). PCMV utilized[2-¹³C]glucose mainlyfor glycolysis, producing[2-¹³C]lactate.Simultaneously, PCMV utilized the glycolytic intermediate [1-¹³C]fructose1,6-bisphosphate (FBP) mainly for gluconeogenesis, producing[1-¹³C]glucose withonly minor[3-¹³C]lactateproduction. The dissimilarity in metabolism of[2-¹³C]FBP derivedfrom [2-¹³C]glucosebreakdown and metabolism of exogenous[1-¹³C]FBPdemonstrates that carbohydrate metabolism is compartmented in PCMV.Because glycolytic enzymes interact with microtubules, we disruptedmicrotubules with vinblastine. Vinblastine treatment significantlydecreased[2-¹³C]lactate peakintensity (87.8 ± 3.7% of control). The microtubule-stabilizing agent taxol also reduced[2-¹³C]lactate peakintensity (90.0 ± 2.4% of control). Treatment with both agentsfurther decreased[2-¹³C]lactateproduction (73.3 ± 4.0% of control). Neither vinblastine, taxol,or the combined drugs affected[1-¹³C]glucose peakintensity (gluconeogenesis) or disrupted the compartmentation ofcarbohydrate metabolism. The similar effects of taxol and vinblastine, drugs that have opposite effects on microtubule assembly, suggest thatthey produce their effects on glycolytic rate by competing withglycolytic enzymes for binding, not by affecting the overall assemblystate of the microtubule network. Glycolysis, but not gluconeogenesis,may be regulated in part by glycolytic enzyme-microtubule interactions.

     

Acute control of fatty acid synthesis by cyclic AMP in the chick liver cell: possible site of inhibition of citrate formation.

Glucagon and N,(6)O(2)-dibutyryl cyclic adenosine 3',5'-cyclic monophosphate (Bt(2)cAMP) inhibit fatty acid synthesis from acetate by more than 90% and prevent citrate formation in chick hepatocytes metabolizing glucose. With substrates that enter glycolysis at or below triose-phosphates, e.g., fructose, lactate, or pyruvate, Bt(2)cAMP has no effect on the citrate level and its inhibitory effect on fatty acid synthesis is substantially reversed. Because acetyl-CoA carboxylase requires a tricarboxylic acid activator for activity, it is proposed that regulation of fatty acid synthesis by Bt(2)cAMP is due, in part, to changes in the citrate level. Reduced citrate formation appears to result from a cAMP-induced inhibition of glycolysis. Bt(2)cAMP inhibits (14)CO(2) production from [1-(14)C]-, [6-(14)C]-, and [U-(14)C]glucose and has little effect on (14)CO(2) formation from [1-(14)C]- or [2-(14)C]pyruvate or from [1-(14)C]fructose. [(14)C]Lactate formation from glucose is depressed 50% by Bt(2)cAMP. In the presence of an inhibitor of mitochondrial pyruvate transport lactate accumulation is enhanced, but continues to be lowered 50% by Bt(2)cAMP. The activity of phosphofructokinase is greatly decreased in Bt(2)cAMP-treated cells while the activities of pyruvate kinase and acetyl-CoA carboxylase are unaffected. It appears that decreased glycolytic flux and decreased citrate formation result from depressed phosphofructokinase activity. Fatty acid synthesis from [(14)C]acetate is partially inhibited by Bt(2)cAMP in the presence of fructose, lactate, and pyruvate despite a high citrate level. Incorporation of [(14)C]fructose, [(14)C]pyruvate, or [(14)C]lactate into fatty acids is similarly depressed by Bt(2)cAMP. Synthesis of cholesterol from [(14)C]acetate or [2-(14)C]pyruvate is unaffected by Bt(2)cAMP. These results implicate a second site of inhibition of fatty acid synthesis by Bt(2)cAMP that involves the utilization, but not the production, of cytoplasmic acetyl-CoA.-Clarke, S. D., P. A. Watkins, and M. D. Lane. Acute control of fatty acid synthesis by cyclic AMP in the chick liver cell: possible site of inhibition of citrate formation.     

Estimation of gluconeogenesis in newborn infants

The rate of glucose turnover (R(a)) and gluconeogenesis (GNG) via pyruvate were quantified in seven full-term healthy babies between 24 and 48 h after birth and in twelve low-birth-weight infants on days 3 and 4 by use of [(13)C(6)]glucose and (2)H(2)O. The preterm babies were receiving parenteral alimentation of either glucose or glucose plus amino acid with or without lipids. The contribution of GNG to glucose production was measured by the appearance of (2)H on C-6 of glucose. Glucose R(a) in full-term babies was 30 +/- 1.7 (SD) micromol. kg(-1). min(-1). GNG via pyruvate contributed approximately 31% to glucose R(a). In preterm babies, the contribution of GNG to endogenous glucose R(a) was variable (range 6-60%). The highest contribution was in infants receiving low rates of exogenous glucose infusion. In an additional group of infants of normal and diabetic mothers, lactate turnover and its incorporation into glucose were measured within 4-24 h of birth by use of [(13)C(3)]lactate tracer. The rate of lactate turnover was 38 micromol. kg(-1). min(-1), and lactate C, not corrected for loss of tracer in the tricarboxylic acid cycle, contributed approximately 18% to glucose C. Lactate and glucose kinetics were similar in infants that were small for their gestational age and in normal infants or infants of diabetic mothers. These data show that gluconeogenesis is evident soon after birth in the newborn infant and that, even after a brief fast (5 h), GNG via pyruvate makes a significant contribution to glucose production in healthy full-term infants. These data may have important implications for the nutritional support of the healthy and sick newborn infant.