Evidence for S-adenosylmethionine independent catabolism of methionine in the rat

Experiments conducted with rats in vivo comparing the metabolism of methionine and S-methyl-L-cysteine and in vitro comparing methionine, S-methyl-L-cysteine and S-adenosyl-L-methionine indicate that a substantial portion of the oxidative metabolism of the methionine methyl group occurs by pathways that are independent of S-adenosylmethionine formation. Inclusion of 1.2% or 2.4% of S-methyl-L-cysteine in a diet containing 3% of L-methionine depressed the conversion of the methionine methyl and carboxyl carbons to CO2 by 39% and 28%, and 52% and 33%, respectively, for the two levels of S-methyl-L-cysteine. Inclusion of 1.65% of methionine in a diet containing 2.4% of S-methyl-L-cysteine did not affect the conversion of the methyl group of S-methylcysteine to CO2, but 3% of methionine depressed the conversion of the S-methylcysteine methyl group to CO2 to 87% of control values. Greater inhibitions were seen when these substrates were compared in a liver homogenate. In a rat liver homogenate system optimized for the conversion of the methyl group of methionine to CO2, the rate of conversion of the methyl group of S-adenosyl-L-methionine to CO2 was less than 1% of that of methionine even when the concentration of S-adenosylmethionine was saturating. Addition of saturating levels of unlabeled S-adenosymethionine to the homogenate system did not effect the rate of conversion of the methionine methyl carbon to CO2. Although S-adenosylmethionine-dependent metabolism of methionine, leading to incorporation of the methyl carbon into sarcosine and serine, could be demonstrated in liver homogenates, essentially all of the CO2 produced from the methionine methyl group was derived by a pathway or pathways independent of S-adenosylmethionine formation. Formaldehyde and formate have been tentatively identified as intermediates in catabolism of the methionine methyl group by this (these) pathway(s).     

Altered metabolism of the methionine methyl group in the leukocytes of patients with schizophrenia

Previous work has indicated that abnormal methylation processes may be associated with schizophrenia. In this study, leukocytes from patients with schizophrenia were incubated with methyl-14C-L-methionine and the evolved 14CO2 measured. With increasing concentration of methionine, the evolved 14CO2 was lower in the patients than in normal control subjects. The incorporation of 14C into protein was the same in both groups, and when carboxyl-14C-L-methionine was used the evolved 14CO2 was the same in both groups, thus excluding the possibility that altered incorporation into protein or oxidation of the methionine molecule as a whole were responsible. The observed differences in methionine-methyl metabolism suggest that an abnormality in transmethylation processes or in oxidation of the methyl group to CO2 is associated with schizophrenia. That this occurs in a peripheral tissue indicates that the abnormality is not restricted to the central nervous system.     

Effect of methionine on the metabolism of formate and histidine by rats fed folate/vitamin B-12-methionine-deficient diet

The metabolism of formate and histidine were compared in rats and in perfused livers of rats on diets deficient in vitamin B-12, methionine, and folic acid. Excretion of formate and formiminoglutamic acid, and the oxidation of [2-14C]histidine and [14C]formate to 14CO2 were measured. Liver folate levels decreased to 40% of normal on the vitamin B-12- and methionine-deficient diets but the rate of oxidation of histidine to CO2 in the whole animal decreased to 15% of normal. This indicated a reduction in the metabolic activity of the liver folates in vitamin B-12deficiency. Comparison of formate and histidine catabolism in folic acid deficiency showed that the oxidation of histine was decreased to 5% of normal but formate oxidation was decreased to only 30% of normal. This indicates that 25% of formate oxidation normally proceeds by a non-folate-dependent pathway.     

Amino acid metabolism in the piglet. 2. Influence of fasting on plasma free amino acid concentration and in vivo oxidation of methionine, isoleucine and threonine

1. The influence of a 24 h fast on the concentrations of free amino acids in the plasma, and upon the oxidation rates of methionine, isoleucine and threonine was studied (using early weaned, 4-week-old piglets which were receiving a semi-purified diet. 2. There was no change in the total concentration of the essential amino acids as a result of the 24 h fast: the concentration of the branched-chain amino acids increased, but the effect of this was offset by decreases in the concentrations of arginine, histidine, lysine, methionine and phenylalanine. There was a reduction in the concentration of the non-essential amino acids. 3. The piglets received infusions of L-[I-14C]methionine, L-[U-14C]isoleucine and L-[U-14C]-threonine, and the recovery of the label in carbon dioxide was determined. Less than 5% of the activity from methionine was recovered in the CO2 from the fed piglets, whereas 12% was recovered from the fasted piglets. The corresponding values with threonine were 11 and 19% but there was no effect of fasting on the recovery of the label from isoleucine in CO2. 4. The initial dilution of a single dose of a labelled amino acid infused into the bloodstream depends on the plasma concentration of the amino acid. Nutritional regimens may effect the free amino acid concentration in the plasma. Thus comparisons based upon direct determination of activity recovered in CO2 from the labelled dose of an amino acid with animals on different nutritional regimens could not misleading, unless the differences in the concentrations of the amino acid in the plasma are considered.     

Folate utilization by monomeric versus heterotetrameric sarcosine oxidases

There are two types of bacterial sarcosine oxidases. The heterotetrameric enzymes contain subunits ranging in size from about 10 to 100 kDa, noncovalently bound FAD and NAD+, and covalently bound FMN attached to the beta subunit (42-45 kDa). Monomeric sarcosine oxidases are similar in size to the beta subunit in the heterotetramers and contain covalently bound FAD. Formaldehyde formation during sarcosine oxidation by several heterotetrameric sarcosine oxidases was suppressed in the presence of 50 microM [6S]-tetrahydrofolate, accompanied by a 25-50% increase in the rate of sarcosine oxidation. In contrast, [6S]-tetrahydrofolate caused only a modest decrease in the rate of formaldehyde production with monomeric sarcosine oxidases (approximately 25%), an effect which was virtually entirely attributable to an accompanying decrease in the rate of sarcosine oxidation. In the presence of 100 microM [6R,S]-tetrahydropteroyltriglutamate [H4Pte(Glu)3], the heterotetrameric enzymes catalyzed the formation of 5,10-methylenetetrahydropteroyltriglutamate [5,10-CH2-H4Pte(Glu)3] at a rate which was 35-60% faster than the rate of sarcosine oxidation in the absence of folate. An apparent Km value of 3.1 microM was estimated for [6S]-H4Pte(Glu)3 with the heterotetrameric corynebacterial sarcosine oxidase. In contrast, slow formation of 5,10-CH2-H4Pte(glu)3 was detected during sarcosine oxidation with monomeric sarcosine oxidases, attributable to the nonenzymatic reaction of free formaldehyde with H4Pte(Glu)3. The results show that only the heterotetrameric sarcosine oxidases can use tetrahydrofolates as substrates and, in this regard, they resemble mammalian sarcosine and dimethylglycine dehydrogenases.     

Carbon dioxide stimulates peroxynitrite-mediated nitration of tyrosine residues and inhibits oxidation of methionine residues of glutamine synthetase: both modifications mimic effects of adenylylation

The activity of glutamine synthetase (EC 6.3.1.2) from Escherichia coli is regulated by the cyclic adenylylation and deadenylylation of Tyr-397 in each of the enzyme's 12 identical subunits. The nitration of Tyr-397 or of the nearby Tyr-326 by peroxynitrite can convert the unadenylylated enzyme to a form exhibiting regulatory characteristics similar to the form obtained by adenylylation. The adenylylated conformation can also be elicited by the oxidation of surface-exposed methionine residues to methionine sulfoxide. However, the nitration of tyrosine residues and the oxidation of methionine residues are oppositely directed by the presence and absence of CO2. At physiological concentrations of CO2, pH 7.4, nitration occurs but oxidation of methionine residues is inhibited. Conversely, in the absence of CO2 methionine oxidation is stimulated and nitration of tyrosine is prevented. It was further established that adenylylation of Tyr-397 precludes its nitration by peroxynitrite. Furthermore, nitration of Tyr-326 together with either nitration or adenylylation of Tyr-397 leads to inactivation of the enzyme. These results demonstrate that CO2 can alter the course of peroxynitrite-dependent reactions and serve notice that (i) the reactions have physiological significance only if they are shown to occur at physiological concentrations of CO2 and physiological pH; and (ii) the peroxynitrite-dependent nitration of tyrosine residues or the oxidation of methionine residues of metabolically regulated proteins can seriously compromise their biological function.     

Endogenous loss of leucine and methionine in adult male rats

1. The fractional rate of loss of 14C and body-weight was measured in adult male rats after giving 14C-labelled methionine or leucine and maintaining rats for 30 d on a low-protein or a specific methionine+cystine-free diet: carcasses were then analysed for protein and fat 14C radioactivity. 2. The fractional loss of 14CO2 from [14C]methionine or [14C]leucine between day 20 and day 30 was always greater than the fractional loss of body-weight. 3. Carcass protein 14C radioactivity after giving [14C]leucine was higher than after giving [14C]methionine, but fat 14C radioactivity after either 14C-labelled amino acid was only a small proportion of the total body 14C radioactivity. 4. After correction of the fractional loss of 14CO2 for urinary 14C loss, but not body-weight loss, absolute amino acid loss was calculated using published values for methionine and leucine content of rats. 5. The best estimates of endogenous amino acid loss obtained using I-14C-labelled amino acids, expressed as mg/kg body-weight 0.75 per day were leucine 79, methionine 38.     

The continuous kinetic determination of p-nitroanisole O-demethylation in hemoglobin-free perfused rat liver

P-Nitroanisole O-demethylation by perfused rat liver based on the spectral properties of the product, p-nitrophenolate, was determined continuously. Rates of p-nitrophenol production in this system were sensitive to inhibition by CO. p-Nitrophenolate production by livers of normal animals was linear for up to 30 minutes; however, rates were only linear for 1 to 2 minutes followed by a steady decline in induced (6-fold) livers from phenobarbital-treated rats. Only a small portion (24%) of this steady decline could be accounted for by the formation of conjugation products. Additionally, infusion of p-nitrophenol (14 micronM) was not associated with a decline in rate. The decline in rate in induced livers was reversed by glucose, suggesting that an intimate relationship may exist between drug and carbohydrate metabolism in the intact liver. Alteration in rates of p-nitroanisole metabolism with various inducing agents of the mixed-function oxidation system (phenobarbital; ethanol) produced parallel changes in rates of hepatic lactate production, most likely reflecting the aciton of p-nitrophenol to uncouple oxidative phosphorylation. The data support the hypothesis that the decline in rate in p-nitroanisole O-demethylation in livers from phenobarbital-treated rats is due to reduced availability of NADPH for mixed-function oxidation.     

Biodegradability of [14C]methylcellulose by activated sludge

Three Methocel methylcellulose ethers of 1.9 degree of substitution with [14C]methyl labels were shown to be biodegradable using batch-type activated sludge tests. The maximum rate for conversion to 14CO2, attained after 1 week, was only 0.62 mg of methylcellulose/g of mixed liquor volatile solids per day. In 20 days, 55 to 73% of the radioactivity had been removed from solution as 14CO2, and the suspended solids contained 12 to 15% of the original radioactivity. Only 4% of the original methylcellulose appeared to be polymeric after the 20-day period. Thin-layer chromatography of supernatant liquid indicated at least two degradation products.     

Surgical treatment of Ebstein''s anomaly

Up to 88% of a single dose of methyl[14C]methacrylate in rats is expired as 14CO2 in 10 days (65% in 2 h), irrespective of the route of administration and of the specific labelling of the propylene residue of the molecule. The implications of this observation, and of the excretion of small amounts of [14C]methylmalonate, [14C]-succinate and probably of [14C]beta-hydroxyisobutyrate and 2-formylpropionate, and of the formation of [14C] normal, physiological metabolites that may be accounted for by anabolism both from 14CO2 and from [14C]acetate emergent from the citric acid cycle, are that the metabolic pathway concerned involves intermediary metabolism and relates to mitochondrial function. Present findings are discussed in relation to the imputations of a report of carcinogenic risk.     

A comparative study of formaldehyde and carbon monoxide complete oxidation on MnOx-CeO2 catalysts

MnOx-CeO2 composite catalysts were prepared by a coprecipitation method and tested for formaldehyde (HCHO) and carbon monoxide (CO) oxidation. X-ray photon spectroscopy (XPS) results indicated that the average oxidation state of surface Mn species in CeMn composite catalyst was higher compared to the pure MnOx. The enhancement of reactivity for HCHO oxidation was due to the activation of the lattice oxygen species in MnOx by the addition of CeO2, which was confirmed by the H2 temperature programmed reduction (H2-TPR) results. The remarkable enhancement of reactivity for CO oxidation by the addition of CeO2 was due to the active oxygen species generated on the CeO2 surface which directly participated in the reaction.     

Growth depression and tissue reaction to the consumption of excess dietary methionine and S-methyl-L-cysteine

Similar depressions in growth were observed when rats consumed a 10% casein basal diet containing equal quantities of either methionine or S-methyl-L-cysteine. Supplemental glycine or serine partially alleviated the growth depression caused by the high levels of methionine but were ineffective in alleviating the growth depression caused by high levels of S-methylcysteine. Histological examination of five organs of rats fed the basal, high methionine or high S-methylcysteine diet for 6, 13 or 20 days revealed that only the spleens were affected in that there was erythrocyte engorgement and an accumulation of hemosiderin. The intensity of iron staining in spleens decreased from the second to the third week. The similarity in the depression of growth and splenic damage observed in rats consuming high levels of methionine or S-methylcysteine is consistent with an earlier suggestion that metabolism of the methionine or S-methylcysteine is consistent with an earlier suggestion that metabolism of the methyl group is in some way involved in the toxicity of methionine.     

Regulatory factors associated with synthesis of the osmolyte glycine betaine in the halophilic methanoarchaeon Methanohalophilus portucalensis

The halophilic methanoarchaeon Methanohalophilus portucalensis can synthesize de novo and accumulate beta-glutamine, Nepsilon-acetyl-beta-lysine, and glycine betaine (betaine) as compatible solutes (osmolytes) when grown at elevated salt concentrations. Both in vivo and in vitro betaine formation assays in this study confirmed previous nuclear magnetic resonance 13C-labelling studies showing that the de novo synthesis of betaine proceeded from glycine, sarcosine, and dimethylglycine to form betaine through threefold methylation. Exogenous sarcosine (1 mM) effectively suppressed the intracellular accumulation of betaine, and a higher level of sarcosine accumulation was accompanied by a lower level of betaine synthesis. Exogenous dimethylglycine has an effect similar to that of betaine addition, which increased the intracellular pool of betaine and suppressed the levels of Nepsilon-acetyl-beta-lysine and beta-glutamine. Both in vivo and in vitro betaine formation assays with glycine as the substrate showed only sarcosine and betaine, but no dimethylglycine. Dimethylglycine was detected only when it was added as a substrate in in vitro assays. A high level of potassium (400 mM and above) was necessary for betaine formation in vitro. Interestingly, no methylamines were detected without the addition of KCl. Also, high levels of NaCl and LiCl (800 mM) favored sarcosine accumulation, while a lower level (400 mM) favored betaine synthesis. The above observations indicate that a high sarcosine level suppressed multiple methylation while dimethylglycine was rapidly converted to betaine. Also, high levels of potassium led to greater amounts of betaine, while lower levels of potassium led to greater amounts of sarcosine. This finding suggests that the intracellular levels of both sarcosine and potassium are associated with the regulation of betaine synthesis in M. portucalensis.     

The role of the locus coeruleus and N-methyl-D-aspartic acid (NMDA) and AMPA receptors in opiate withdrawal

The turnover rates of plasma lactate, normalized for O2 consumption rate, are higher in the fetus than in the adult. This occurs despite very low rates of fetal gluconeogenesis which preclude the recycling of lactate carbon into glucose. In an effort to establish the main routes of disposal of fetal plasma lactate, 12 midgestation ovine fetuses (age 74 +/- 1 days) were infused intravenously at constant rate with L-[U-14C]lactate for a 4-hour period. At the end of the infusion, the amounts of 14C retained by the fetus and by the placenta, and the distribution of the retained 14C in free and protein-bound amino acids and in lipids were measured. Of the total 14C infused, 17.0 +/- 1.4% was recovered in the placenta, 4.0 +/- 0.3% in the fetal liver, and 15.0 +/- 0.8% in the extrahepatic fetal tissues. Of the retained radioactive carbon, 45-57% was recovered in the free and protein-bound amino acid fractions and 11-17% in the lipid fractions. Approximately 90% of the 14C in the free amino acid fractions was present as glutamate/glutamine, serine, glycine, and alanine carbon. In conjunction with data on fetal CO2 production from lactate carbon, these results demonstrate that the main routes of fetal lactate disposal are oxidation and synthesis of nonessential amino acids and lipids.     

Clinical diagnosis with the stable isotope 13C in CO2 breath tests: methodology and fundamental considerations

The methodology for measuring in vivo oxidation of substrates labeled with the nonradioactive carbon isotope 13C has been developed with isotope ratio mass spectrometry. The use of 13C offers the possibility of utilizing CO2 breath tests in infants, children, pregnant women, and all subjects in whom 14CO2 breath tests cannot be used. The excretion of 140 nmol/kg-hr of 13CO2 produced from the oxidation of the labeled substrate could be detected with 95% confidence during a total CO2 excretion of 9 mM/kg-hr. The precision of CO2 breath tests using 13C is limited by the natural fluctuations of the ratio of 13C/12C in expired CO2, which occur with a standard deviation of 0.72%, or approximately 7 parts 13CO2 per 10(6) parts expired CO2. Larger excursions in the ratio were observed if the subjects ate shortly before or during the breath test. Clinically significant diagnostic tests can reasonably be expected to require the excretion of 2 to 20 times as much labeled CO2, or 0.28 to 1.4 micronM/kg-hr.     

Metabolism of [1-(14)C] and [2-(14)C] leucine in cultured skin fibroblasts from patients with isovaleric acidemia. Characterization of metabolic defects

Leucine metabolism in cultured skin fibroblasts from patients with isovaleric acidemia was compared with that in normal fibroblasts and in cells from patients with maple syrup urine disease using [1-(14)C] and [2-(14)C] leucine as substrates. Inhibitory effects of methylenecyclopropylacetic acid on leucine metabolism in normal cells were also investigated. Production of 14CO2 from [2-(14)C] leucine was very reduced (96-99%) in both types of mutant cells. Radioactive isovaleric acid accumulated in assay media with isovaleric acidemia cells but not in those with maple syrup urine disease cells. Unexpectedly, 14CO2 production from [1-(14)C] leucine was partially depressed (80%) in isovaleric acidemia cells whereas in maple syrup urine disease cells it was strongly depressed (99%) as expected. These two mutant cells were clearly distinguished by detection of 14C-isovaleric acid accumulation after incubation with [2-(14)C] leucine. A pattern of inhibition of leucine oxidation similar to that seen in isovaleric acidemia cells was induced in normal cells by the addition of 0.7 mM methylenecyclopropylacetic acid to the assay medium. The partial inhibition of [1-(14)C] leucine oxidation seen in isovaleric acidemia cells and also in normal cells in the presence of the inhibitor appears to be, at least in part, due to an accumulation of isovalerate in the cells. Isovaleric acid (5-10) mM) inhibited [1-(14)C] leucine oxidation 32-68% when added to the assay medium with normal cells. Addition of flavin adenine dinucleoside to culture medium or assay medium or both did not restore oxidation of either leucine substrate in isovaleric acidemia cells.     

The determination of methionine in proteins by gas-liquid chromatography

Intact methionine residues in proteins were rapidly and precisely determined by measuring methyl thiocyanate released during the reaction with CNBr and separated by g.l.c. Conditions for the reaction and for chromatography on columns of Porapak P-S are described. The recovery of methyl thiocyanate from several methionine derivatives and analogues were examined. Carbamoylmethionine was adopted as a stable primary standard and ethyl thiocyanate as internal standard. The measured methionine content of several isolated proteins was close to the theoretical value indicated by previous work and the results for these and a range of food proteins agreed well with results obtained by ion-exchange chromatography after performic acid oxidation. Since CNBr does not react with methionine sulphoxide and a preliminary hydrolysis is not required, the method discriminates between methionine and any methionine sulphoxide that may be present. It could be useful in studies on the nutritional availability of methionine in processed foods.     

Marginal irregularity of flat elevated type of colorectal tumor as a marker of malignant potential

The open reading frame yjbR which had been sequenced as a part of the Bacillus subtilis genome project encodes a putative 40.9-kDa protein. The yjbR-coding sequence was slightly similar to those of bacterial sarcosine oxidases and possibly compatible with the tertiary structure of the porcine kidney D-amino acid oxidase. The yjbR gene product was overproduced in Escherichia coli, purified to homogeneity from the recombinant strain, and characterized. This protein effectively catalyzed the oxidation of sarcosine (N-methylglycine), N-ethylglycine and glycine. Lower activities on D-alanine, D-valine, and D-proline were detected although no activities were shown on L-amino acids and other D-amino acids. Since glycine is a product and not a substrate for sarcosine oxidase, this protein is not a type of demethylating enzymes but a novel deaminating oxidase, named glycine oxidase as a common name. Several enzymatic properties of the B. subtilis glycine oxidase were also investigated.     

Biodegradation of the gasoline oxygenates methyl tert-butyl ether, ethyl tert-butyl ether, and tert-amyl methyl ether by propane-oxidizing bacteria

Several propane-oxidizing bacteria were tested for their ability to degrade gasoline oxygenates, including methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME). Both a laboratory strain and natural isolates were able to degrade each compound after growth on propane. When propane-grown strain ENV425 was incubated with 20 mg of uniformly labeled [14C]MTBE per liter, the strain converted > 60% of the added MTBE to 14CO2 in < 30 h. The initial oxidation of MTBE and ETBE resulted in the production of nearly stoichiometric amounts of tert-butyl alcohol (TBA), while the initial oxidation of TAME resulted in the production of tert-amyl alcohol. The methoxy methyl group of MTBE was oxidized to formaldehyde and ultimately to CO2. TBA was further oxidized to 2-methyl-2-hydroxy-1-propanol and then 2-hydroxy isobutyric acid; however, neither of these degradation products was an effective growth substrate for the propane oxidizers. Analysis of cell extracts of ENV425 and experiments with enzyme inhibitors implicated a soluble P-450 enzyme in the oxidation of both MTBE and TBA. MTBE was oxidized to TBA by camphor-grown Pseudomonas putida CAM, which produces the well-characterized P-450cam, but not by Rhodococcus rhodochrous 116, which produces two P-450 enzymes. Rates of MTBE degradation by propane-oxidizing strains ranged from 3.9 to 9.2 nmol/min/mg of cell protein at 28 degrees C, whereas TBA was oxidized at a rate of only 1.8 to 2.4 nmol/min/mg of cell protein at the same temperature.     

Estimates of glycolysis, pyruvate (de)carboxylation, pentose phosphate pathway, and methyl succinate metabolism in incapacitated pancreatic islets

Pancreatic islets were cultured for 24 h in the presence of 1 mM glucose, which renders islets incapable of responding to glucose with insulin release. These islets were compared to islets maintained at 20 mM glucose for 24 h. Detritiation of [2-3H]glucose and [5-3H]glucose in 1 mM glucose islets was normal, suggesting that glucose transport and phosphorylation and all enzymes of glycolysis were not down-regulated in the incapacitated islets. 14CO2 formation from [U-14C]glucose and [6-14C]glucose was inhibited up to 80% and 14CO2 from methyl succinate was inhibited up to 60%, indicating that down-regulation at (a) mitochondrial site(s) might explain the incapacitated insulin release. 14CO2 formation from [3,4-14C]glucose (which becomes [1-14C]pyruvate) was decreased, indicating that the reaction catalyzed by pyruvate dehydrogenase was down-regulated. This decrease, however, was not as large as the decreases in 14CO2 formation from [U-14C]glucose, [2-14C]glucose (which becomes [2-14C]pyruvate), or [6-14C]glucose (which becomes [3-14C]pyruvate), indicating that other reactions were also down-regulated. 14CO2 formation from [1-14C]glucose was inhibited less than that from [6-14C]glucose in the incapacitated islets (34 vs 54%) and these rates indicated that flux of glucose through the pentose phosphate pathway was increased in the incapacitated islet, such that 29% (0.4 nmol of 1.4 glucose/100 islets/90 min) was metabolized via this pathway in the incapacitated islet but only 3.4% (0.1 of 2.9 nmol glucose/100 islets/90 min) was metabolized via the pentose pathway in the 20 mM glucose islets. With rates of 14CO2 evolved from glucose labeled at C2 and C6 and from methyl succinate labeled at C1 + C4 and C2 + C3 the 14CO2 ratio formula was used to calculate the ratios of carboxylated and decarboxylated pyruvate. Roughly equal amounts of pyruvate entered the citric acid cycle by each route in islets maintained for 24 h at 1, 5, or 20 mM glucose. The results indicate that decarboxylation and carboxylation of pyruvate were about equally suppressed in incapacitated islets and that direct inhibition of reactions of the cycle was unlikely. This is consistent with evidence which indicates that down-regulation of both pyruvate carboxylase and pyruvate dehydrogenase occurs in incapacitated islets, i.e., under long-term conditions that modify amounts of enzymes (MacDonald et al., 1991, J. Biol. Chem. 266, 22392-22397).(ABSTRACT TRUNCATED AT 400 WORDS)