A. L. Greenbaum

The distribution of hepatic metabolites and the control of the pathways of carbohydrate metabolism in animals of different dietary and hormonal status

Abstract A method is described by which the cytoplasmic and mitochondrial content of malate, oxaloacetate, aspartate, glutamate, 2-oxoglutarate, isocitrate, and citrate can be calculated. The values so obtained confirm that oxaloacetate occurs mainly in the cytosol. Aspartate, glutamate, and 2-oxoglutarate appear to be mainly located in the cytosol. Considerable redistribution of these metabolites occurs in the different nutritional and hormonal states. The redox state of the nicotinamide nucleotides in the two compartments has been calculated using the compartmented values. The mitochondrial redox state of the NADP couple appears to be far more reduced than has hitherto been thought. Control of the glycolytic pathway is vested in phosphofructokinase, pyruvate kinase, and glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase. The most important modifier of hepatic phosphofructokinase seems to be fructose-6-phosphate, which may act by changing the K i ; for citrate, thus permitting a sufficient concentration of citrate to be present in the cytosol for fatty acid synthesis without inhibition of phosphofructokinase. This overcomes the difficulty of the requirement for a rapid glycolytic flux simultaneously with lipid synthesis from citrate. Ultimate control of glycolysis may rest with glucokinase. The extent of deviation of triose phosphate isomerase from equilibrium is suggested as an index of glycolytic pathway flux and direction. Compartmentation of metabolites in the span pyruvate to phosphoenolpyruvate provided additional evidence for an increased flux through the control enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase in gluconeogenesis. The possibility that cAMP may be a positive effector of phosphoenolpyruvate carboxykinase is considered. The source of reducing equivalents for gluconeogenesis is examined. It is concluded that transfer of carbon occurs both as malate and aspartate, and that the requirement for reducing equivalents is met in part by the transfer of malate to the cytosol and in part by NADH generated by the fumarate cycle geared to urea production. Calculations of the compartmentation of tricarboxylic acid cycle intermediates suggested that a very different environment may exist in the mitochondria from that deduced from total cell metabolites. Gluconeogenic situations were characterized by increased mitochondrial 2-oxoglutarate and small changes of oxaloacetate, while the total cell contents of both were substantially decreased. Calculations of mitochondrial oxaloacetate by two independent procedures gave similar values, with no evidence for any significant fall below control level. Control of the tricarboxylic acid cycle by the redox state of the nicotinamide nucleotides and by the acetyl CoA CoA ratio is also discussed. Evidence from mass-action ratios and metabolite profiles point to control of the oxidative segment of the pentose phosphate pathway being located at glucose-6-phosphate dehydrogenase, the role of the NADP + NADPH ratio being emphasized. The advantages of using the reactants of 6-phosphogluconate dehydrogenase in the determination of the cytoplasmic NADP redox state are shown. The close agreement between the two different procedures for the calculation of the compartmentation of such key metabolites as malate and citrate lends credence to the validity of such procedures.

The Functional Significance of the Pentose Phosphate Pathway in Synaptosomes: Protection Against Peroxidative Damage by Catecholamines and Oxidants

Abstract: Catecholamines added in vitro in rat brain synaptosomes activate the decarboxylation of glucose radioactively labelled on carbon 1, suggesting an effective activation of the pentose phosphate pathway. Stimulation also occurred with phenazine methosulphate, reduced glutathione and hydrogen peroxide. The activation of the pentose phosphate pathway by 5‐hydroxytryptamine, noradrenaline and dopamine is ascribed to the activation of monoamine oxidase, producing both the respective biogenic aldehyde and hydrogen peroxide. Evidence is presented that the further metabolism of the aldehyde by aldehyde reductase and the removal of hydrogen peroxide by glutathione peroxidase both release the limitation of N ADP+ availability for the pentose phosphate pathway by leading to the oxidation of NADPH. The relevance of the maintenance of reduced NADP+ on brain is discussed in relation to the metabolism of glutathione and to lipid peroxidation.

Aspects of Carbohydrate Metabolism in Developing Brain

This review considers carbohydrate metabolism in the developing brain, in particular the proportion of glucose metabolized via the pentose phosphate pathway. Although small in amount, this fraction serves a vital role in some aspects of brain function. Evidence is presented that the pentose phosphate pathway subserves different functions as the developing brain progresses through the stages of growth and myelination to full neurological competence. The general aspects considered are the changing patterns of brain enzymes during development; the flux of glucose through the alternative pathways of glucose metabolism in the developing brain; the functional significance of the pentose phosphate pathway; and the regional and functional association of the pentose phosphate pathway activity and the detoxication of biogenic amines.

The role of cyclic nucleotides in the development and function of rat mammary tissue

It has been previously reported that the tissue content of cyclic AMP in rat mammary gland rises continuously to the end of pregnancy and then falls progessively to its lowest value by the 16th day of lactation [ 11, suggesting that the growth and development of the gland are related to the issue content of cyclic AMP, while the initiation, and scale, of lactation may be related to the decreasing content of the nucleotide. In studies with rat mammary gland explants in culture [2] cyclic AMP inhibited the increase of enzyme activities normally associated with lactogenesis, in particular the activities of those enzymes involved in lipogenesis, and also markedly depressed the synthesis of DNA, RNA and fatty acids. This analogy with the pleiotypic response observed for other cells grown in culture [3], and the reversal by cyclic GMP of the pleiotypic inhibition of 3T3 cells treated with dibutyryl cyclic AMP [4] , led us to investigate the simultaneous fluctuations of both cyclic nucleotides throughout the lactation cycle. differentiation of the mammary gland during pregnancy, its response to hormones and the promotion of specific cell programmes for the synthesis of the characteristic components of milk may all be mediated by modification of the ratio between the two nucleotides.

The effect of di-butyryl cAMP on enzymatic and metabolic changes in explants of rat mammary tissue

Summary The effect of cAMP on the activities of some enzymes and on the rates of synthesis of DNA, RNA, casein and fatty acids in rat mammary gland explants grown in culture has been examined. cAMP inhibits the increase of enzyme activities associated with the development of the tissue under the culture conditions and, in particular, the activities of those enzymes involved in lipogenesis. It also markedly depresses the synthesis of DNA, RNA and fatty acids. Casein synthesis is slightly increased. These results are discussed in relation to the possible role(s) of cAMP as a regulator of mammary gland development and function.

Enzymic differentiation in pathways of carbohydrate metabolism in developing brain

Abstract The entire sequence of enzymes of the pentose phosphate pathway, the glycolytic pathway and certain enzymes of fatty acid synthesis were measured in developing rat brain. Coordinated increases in enzymes of the glycolytic pathway were observed, with apparent discrimination in that greater changes were found among enzymes of low turnover capability (Vmax: Km). During development a sequential pattern emerged with early changes in phosphofructokinase and aldolase followed by triosephosphate isomerase, glycerophosphate dehydrogenase and phosphoglyceromutase; enzymes of the pentose phosphate pathway remained relatively constant, while a marked and parallel fall was seen in acetyl CoA car☐ylase, fatty acid synthetase and NADP+-linked isocitrate dehydrogenase.

Changes of the activities of adenyl cyclase and cAMP-phosphodiesterase and of the level of 3′5′ cyclic adenosine monophosphate in rat mammary gland during pregnancy and lactation

Abstract The activities of adenyl cyclase, the low- and high- K m cAMP phosphodiesterases and the tissue level of cAMP have been measured in mammary glands taken from pregnant and lactating rats. Adenyl cyclase reaches its maximum activity late in pregnancy and thereafter falls continuously throughout lactation. The high- K m phosphodiesterase rises slowly in pregnancy but reaches a maximum late in lactation. The tissue cAMP content is maximal at the end of pregnancy and then falls progressively to its lowest level by the sixteenth day of lactation. These changes are discussed in relation to the metabolic adaptation of the gland and its responsiveness to hormonal stimulation.

Constant and specific proportion groups of enzymes in rat mammary gland and adipose tissue in relation to lipogenesis.

The observation by Pette et al. [l-3] on the existence of constant and specific proportion groups of enzymes and the value of these groups in differentiating enzymes of especial importance in the individual metabolism of different tissues has prompted a number of studies in a variety of tissues which attempt to specify enzymes of particular significance to those tissues [4-71. This principle has been extended to investigate a single tissue under different functional conditions and it has been applied here to the changes of enzyme content found in the mammary gland as the tissue progresses from the relatively quiescent state found in late pregnancy to the highly active state characteristic of the peak of lactation. For comparison, the relative enzyme changes have also been recorded for adipose tissue in conditions of normal, depressed and hyperlipogenic states. This comparison is a relevant one in that both tissues have metabolic patterns largely directed to fatty acid synthesis although it may be noted that, while the mammary gland is an open-ended secretory system and is virtually a unidirectional biosynthetic tissue, adipose tissue may, at different times, act either as a lipogenie tissue or as a fatty acid exporter. In the mammary gland there is a marked increase in enzymic activity from pregnancy to the height of lactation. When these activities are expressed relative to that of the rate-limiting enzyme of the glycolytic sequence (namely PFK) a distinct pattern emerges,

Thyroid hormone control of cyclic nucleotide phosphodiesterases and the regulation of the sensitivity of the liver to hormones

One of the more striking, but least studied, aspects of thyroid action is its effect on the hormone responsiveness of tissues. The action of lipolytic hormones such as glucagon, catecholamines and ACTH is markedly reduced in adipose tissue following thyroidectomy, while the action of insulin is potentiated after administration of thyroid hormone [l] . Insulin and glucagon both modulate the cyclic nucleotide content of liver and adipose tissue, insulin causing a rapid and transient rise in cGMP [2,3] and glucagon a marked rise of CAMP [4,5] . The reports that there is an increase of CAMPand cGMP-phosphodiesterases in adipose tissue following thyroidectomy [6-91 may well be particularly relevant to these tissue responses to hormones. The similarities in the response to thyroidectomy of the enzymes of carbohydrate metabolism and of fatty acid synthesis in rat liver and adipose tissue [lo] prompted the present study of the enzymes regulating the tissue level of the cyclic nucleotides in rat liver. Changes in the balance of the enzymes adjusting the tissue content of these nucleotides could provide a common locus for thyroid action in a number of tissues responsive to insulin and glucagon and thus act as a unifying factor in the coordination of liver and adipose tissue metabolism.

The effect of quinolinic acid on the content and distribution of hepatic metabolites

Abstract The effect of quinolinic acid treatment on the hepatic metabolite profile and the flux of glucose through the alternative pathways of metabolism have been measured, and the distribution of metabolites between the cytosolic and mitochondrial compartments has been calculated. Marked increases of the total-cell polycarboxylic anions were found and these were, in order of magnitude: malate, citrate, isocitrate, aspartate, 2-oxoglutarate, and glutamate. Calculation of the compartmented values suggested that the major increase was in the mitochondrial compartment: cytosolic glutamate, 2-oxoglutarate, and oxaloacetate were decreased and only aspartate increased in this compartment. The changes of the mitochondrial/cytosolic anion ratio was most marked, 60-fold, in the case of 2-oxoglutarate. It is suggested that inhibition of transport of 2-oxoglutarate by quinolinic acid could, by blocking the operation of the aspartate shuttle, contribute to the inhibition of gluconeogenesis from lactate. Metabolite and flux data suggest an increase in the rate of lipogenesis in quinolinic acid-treated rats with the decrease of long-chain acyl CoAs, caused by this treatment, being the possible effector for this activation.