Impairment of hepatic pyruvate metabolism in phenylketonuria

In patients with untreated classical phenylketonuria, elevated plasma levels of pyruvate, lactate, phenylalanine and phenylpyruvate were observed. After about 10 days on a low-phenylalanine diet, the levels of pyruvate, lactate and phenylpyruvate in plasma of treated patients returned to normal; the concentrations of phenylalanine in plasma were markedly lowered. In plasma from hyperphenylalaninemic subjects, phenylpyruvate was not detectable; pyruvate and lactate were within normal limits. Phenylpyruvate at a concentration of about 1 mM inhibited pyruvate carboxylation by human and rat liver homogenates by about 50%; phenylalanine had no effect on this process. The values of apparent Km for pyruvate and Ki for phenylpyruvate of human liver pyruvate carboxylase were approximately 0.27 mM and 1.4 mM, respectively. These studies suggest an impairment in hepatic pyruvate metabolism in untreated phenylketonuric patients.     

The effects of adenine nucleotides on pyruvate metabolism in rat liver

1. The effects of adenine nucleotides on pyruvate metabolism by isolated liver cells and isolated mitochondria have been investigated. The amount of pyruvate carboxylated has been estimated by determining the tricarboxylic acid-cycle intermediates, glutamate and aspartate accumulating in the incubation medium. The extent of pyruvate oxidation has been assessed by measuring oxygen uptake and the yield of ¹⁴CO₂ from [1-¹⁴C]pyruvate and [2-¹⁴C]pyruvate. 2. When catalytic amounts of adenine nucleotides (1–2mm) were added to suspensions of isolated liver cells incubated with pyruvate an ATP:ADP ratio greater than 6:1 was maintained. Both pyruvate oxidation to acetyl-CoA and the oxidation of acetyl-CoA through the tricarboxylic acid cycle were stimulated but pyruvate carboxylation was not affected. The production of acetyl-CoA exceeded the capacity of the cells for the oxidation of acetyl-CoA and the excess was converted into ketone bodies. 3. If a low ATP:ADP ratio was maintained in isolated cells or mitochondria by incubating them with dinitrophenol or hexokinase, pyruvate carboxylation was grossly inhibited, oxygen uptake depressed and ketone-body formation stimulated. Measurement of oxaloacetate concentrations confirmed that under these conditions oxaloacetate was rate-limiting for the oxidation of acetyl-CoA via the tricarboxylic acid cycle. The inclusion in the incubation medium of fumarate (1·25mm) completely prevented the ketogenic action of dinitrophenol or hexokinase. 4. When ADP (5mm) was added to a suspension of isolated liver cells incubated with pyruvate an actual ADP concentration of about 1mm was attained. This brought about effects on pyruvate metabolism similar to those obtained with dinitrophenol or hexokinase. 5. These results support the concept that the relative concentrations of adenine nucleotides within the liver cell may play a role in governing the rates of pyruvate oxidation and carboxylation. In addition, they provide further evidence that the availability of oxaloacetate in the liver cell can play a key role in determining whether acetyl-CoA arising from pyruvate is oxidized through the tricarboxylic acid cycle or converted into ketone bodies.     

The effects of vitamin A deficiency on hepatic folate metabolism in rats

The effects of severe vitamin A deficiency (liver retinol less than 2 micrograms/g) on hepatic folate metabolism in rats were studied. The oxidation of a [ring-2-14C] histidine load or a [14C]formate load to 14CO2 was significantly depressed in vitamin A-deficient rats and those given histidine also excreted more urinary formiminoglutamic acid (FiGlu) than pair-fed controls. The increase in FiGlu excretion was not due to augmented production from histidine, implicating an impairment of FiGlu catabolism. FiGlu formiminotransferase activity was unaltered in vitamin A-deficient rats, but hepatic tetrahydrofolic acid (THF) concentration was decreased by 58% in vitamin A-deficient rats given a histidine load while 5-methyl-THF concentration was increased by 39%. Formyl-THF and total folate levels were similar to controls. A redistribution of folate coenzymes was not found in vitamin A-deficient rats not force fed histidine. A 43% decrease in 10-formyl-THF dehydrogenase activity, which generates both THF and the 14CO2 from the labeled substrates, and an 81% increase in 5,10-methylene-THF reductase activity, which generates 5-methyl-THF, were found in vitamin A-deficient rats. It appears that the production of severe vitamin A deficiency results in selective changes in the activities of hepatic folate-dependent enzymes, so that when a load of a one-carbon donor is given, THF concentration decreases and metabolism of the load is impaired.     

The effect of phenylpyruvate on pyruvate metabolism in rat brain

1. The effect of phenylalanine and phenylpyruvate on the metabolism of pyruvate by isolated mitochondria from rat brain was investigated. 2. Phenylpyruvate inhibited the fixation of H(14)CO(3) (-) in the presence of pyruvate by intact rat brain mitochondria, whereas phenylalanine and other metabolites of this amino acid had no inhibitory effect on this process. 3. Pyruvate carboxylase activity in freeze-dried rat brain mitochondrial preparations was also inhibited only by phenylpyruvate, and a ;mixed type' inhibition was observed. 4. The K(m) for pyruvate of rat brain pyruvate carboxylase was about 0.2mm. 5. The concentration of phenylpyruvate required for a 50% inhibition of H(14)CO(3) (-) fixation by the intact mitochondria and of pyruvate carboxylase activity was dependent on the concentration of pyruvate used in the incubation medium. 6. The possible significance of inhibition of pyruvate carboxylase activity by phenylpyruvate in the brains of phenylketonuric patients is discussed.     

Acute effects of tumor-promoting phorbol esters on hepatic intermediary metabolism

In hepatocytes isolated from meal-fed rats, phorbol 12-myristate 13-acetate as well as phorbol 12,13-didecanoate stimulated de novo fatty acid synthesis in a dose-dependent manner. Moreover, phorbol 12-myristate 13-acetate inhibited ketogenesis from exogenous oleate, but slightly enhanced oleate esterification. The stimulation of esterification was more pronounced with endogenously synthesized fatty acids. In hepatocytes from 24h-starved rats a moderate stimulation of gluconeogenesis and ureogenesis was observed with glutamine as substrate. It is concluded that tumor-promoting phorbol esters mimick the short-term effects of insulin on hepatic fatty acid metabolism.     

Rate-limiting steps for hepatic gluconeogenesis. Mechanism of oxamate inhibition of mitochondrial pyruvate metabolism

Oxamate, structural analog of pyruvate, inhibits gluconeogenesis from pyruvate or substrates yielding pyruvate. The inhibitory effect is the result of a decreased mitochondrial pyruvate utilization. Although the inhibition of gluconeogenesis is competitive for pyruvate, in isolated mitochondria oxamate displays a mixed type kinetics inhibitory pattern of pyruvate utilization. Evidence is presented indicating that this mixed type pattern of inhibition is the result of the action of oxamate on two different sites: noncompetitive inhibition of pyruvate carboxylation, and competitive inhibition of pyruvate entry into the mitochondria. At concentrations of pyruvate above 0.4 mM, although pyruvate carboxylation is decreased by 40% by oxamate, no detectable effects on the gluconeogenic flux were observed. This finding strongly indicates that pyruvate carboxylase is not an important rate-limiting step for hepatic gluconeogenesis. Thus, the inhibition of gluconeogenesis at low pyruvate concentrations (less than 0.4 mM) seems to be the result of an interaction of oxamate with the mitochondrial pyruvate translocator, indicating that pyruvate transport across the mitochondrial membrane is the first nonequilibrium step in the gluconeogenic pathway when low physiological concentrations of this substrate are utilized.     

Early effects of excessive retinol intake on hepatic glycogen metabolism

Oral adminstration of 30,000 IU of retinol once daily for 2-days caused deposition of glycogen in the liver with a concurrent stimulation of hepatic glycogen synthesis, as evidenced by increased in vivo incorporation of d-[U-¹⁴C]glucose into glycogen and increased net synthesis of the polysaccharide in response to feeding of glucose to 20-h fasted rats. Excessive intake of the vitamin increased markedly the activity of glycogen synthetase a and decreased that of phosphorylase. However, feeding of similar doses of retinol to bilaterally adrenalectomized rats failed to cause appreciable deposition of glycogen in the liver and the usual increase in the activity of glycogen synthetase a. Likewise, treatment of rats with actinomycin D blocked the deposition of glycogen in the liver and the increase in the activity of glycogen synthetase a. Adrenalectomy and actinomycin D, however, did not affect the accumulation of retinol in the liver. The adrenals appear to be, directly or indirectly, required for the manifestations of the effects of retinol on the hepatic glycogen metabolism.     

Gluconeogenesis in the cow. The effects of a glucocorticoid on hepatic intermediary metabolism

1. The hepatic concentrations of the ketone bodies and of metabolites and activities of enzymes involved in gluconeogenesis were measured in healthy lactating and non-lactating cows 48h after administration of Voren, an ester of dexamethasone, and compared with those found in control animals given saline. Parallel measurements were also made of the blood concentrations of several of the metabolites. 2. Blood glucose concentrations were raised in the Voren-treated animals, whereas blood ketone body and free fatty acid concentrations were unaltered. Similarly there was no change in the hepatic concentrations of the ketone bodies. 3. Significant increases were found in the hepatic concentrations of citrate, 2-oxo-glutarate and malate in both groups of animals given Voren. 4. The hepatic concentrations of those glycolytic intermediates that were measured either decreased or did not change after Voren treatment. 5. The enzymes aspartate transaminase and fructose 1,6-diphosphatase were unchanged in activity after Voren administration, whereas phosphopyruvate carboxylase (EC 4.1.1.32) activity was depressed in the lactating group. However, glucose 6-phosphatase, tryptophan oxygenase and tyrosine aminotransferase increased in activity. 6. In several cases those hepatic metabolites that increased in concentration after Voren administration were present in lower concentration in normal lactating cows than in normal non-lactating cows. The same applied mutatis mutandis to those metabolites that were decreased by Voren. 7. These findings are discussed in relation to the use of glucocorticoids in the treatment of bovine ketosis.     

Brain metabolism of exogenous pyruvate

Pyruvate given in large doses may be neuroprotective in stroke, but it is not known to what degree the brain metabolizes pyruvate. Intravenous injection of [3-13C]pyruvate led to dose-dependent labelling of cerebral metabolites so that at 5 min after injection of 18 mmoles [3-13C]pyruvate/kg (2 g sodium pyruvate/kg), approximately 20% of brain glutamate and GABA were labelled, as could be detected by 13C nuclear magnetic resonance spectrometry ex vivo. Pyruvate, 9 mmoles/kg, was equivalent to glucose, 9 mmoles/kg, as a substrate for cerebral tricarboxylic acid (TCA) cycle activity. Inhibition of the glial TCA cycle with fluoroacetate did not affect formation of [4-13C]glutamate or [2-13C]GABA from [3-13C]pyruvate, but reduced formation of [4-13C]glutamine by 50%, indicating predominantly neuronal metabolism of exogenous pyruvate. Extensive formation of [3-13C]lactate from [2-13C]pyruvate demonstrated reversible carboxylation of pyruvate to malate and equilibration with fumarate, presumably in neurones, but anaplerotic formation of TCA cycle intermediates from exogenous pyruvate could not be detected. Too rapid injection of large amounts of pyruvate led to seizure activity, respiratory arrest and death. We conclude that exogenous pyruvate is an excellent energy substrate for neurones in vivo, but that care must be taken to avoid the seizure-inducing effect of pyruvate given in large doses.