Patricia McLean

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 Effect of an Aldose Reductase Inhibitor (Sorbinil) on the Level of Metabolites in Lenses of Diabetic Rats

This study examined the effect of an aldose reductase inhibitor (Sorbinil, CP 45634,Pfizer, Sandwich, Kent, United Kingdom) on the metabolite profile of the lens during the first week after induction of diabetes with alloxan. The lens content of sorbitol, fructose, glycerol 3-phosphate, and glucose 6-phosphate was, respectively, 0.33 ± 0.03, 0.55 ± 0.05, 0.10± 0.01, and 0.074 ± 0.006 μmol/g (means ± SEM) in the control group rising to 12.2 ± 0.52, 3.20 ± 0.10, 0.76 ± 0.10, and 0.200 ± 0.009 in lenses from alloxan-diabetic rats. Sorbinil treatment (40 mg/kg) decreased the lens content of sorbitol to 0.60 ± 0.06, fructose to 0.85 ± 0.08, and glycerol 3-phosphate to 0.36 ± 0.03 μmol/g; glucose 6-phosphate remained unchanged. Significantly, the lens content of glutathione was decreased to 60% of the normal value in the diabetic group, but was sustained at normal levels with Sorbinil treatment. The ATP content of the lens was not altered by diabetes or Sorbinil treatment at this time interval. Sorbinil has no significant effect on the above metabolites in the normal rat lens. The effect of Sorbinil in restoring normal levels of glutathione and glycerol 3-phosphate may be a potentially important facet of the action of this drug. The interlocking of metabolic pathways by the redox state of NAD+ /NADH and NADP+/NADPH, their derangement in diabetes, and the wider effects of Sorbinil on the network of reactions in the lens are discussed.

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.

A metabolic and functional overview of brain aging linked to neurological disorders

Close correlations have recently been shown among the late onset complications encountered in diabetes and aging linked to neurobiological disorders. Aging in females and males is considered as the end of natural protection against age related diseases like osteoporosis, coronary heart disease, diabetes, Alzheimer’s disease and Parkinson’s disease, dementia, cognitive dysfunction and hypernatremia. Beside the sex hormones other hormonal changes are also known to occur during aging and many common problems encountered in the aging process can be related to neuroendocrine phenomena. Diabetes mellitus is associated with moderate cognitive deficits and neurophysiologic and structural changes in the brain, a condition that may be referred to as diabetes encephalopathy; diabetes increases the risk of dementia especially in the elderly. The current view is that the diabetic brain features many symptoms that are best described as accelerated brain aging. This review presents and compares biochemical, physiological, electrophysiological, molecular, and pathological data from neuronal tissue of aging and hormone treated control and diabetic animals to arrive at the similarities among the two naturally occuring physiological conditions. Animal models can make a substantial contribution to understanding of the pathogenesis, which share many features with mechanism underlying brain aging. By studying the pathogenesis, targets for pharmacology can be identified, finally leading to delay or prevention of these complications. Antiaging strategies using hormone therapy, chemical and herbal compounds were carried out for reversal of aging effects. Neuronal markers have been presented in this review and similarities in changes were seen among the aging, diabetes and hormone treated (estrogen, DHEA and insulin) brains from these animals. A close correlation was observed in parameters like oxidative stress, enzyme changes, and pathological changes like lipofuscin accumulation in aging and diabetic brain.

Alternative pathways of glucose utilization in brain. Changes in the pattern of glucose utilization in brain during development and the effect of phenazine methosulfate on the integration of metabolic routes.

Abstract The activities of alternative pathways of glucose metabolism in developing rat brain were evaluated by measurement of the yields of 14CO2 from glucose labeled with 14C on carbons 1, 2, 3 + 4, 6 and uniformly labeled glucose, from the detritiation of [2-3H]glucose and from the incorporation of 14C from specifically labeled glucose into lipids by brain slices from cerebral hemispheres and cerebellum. The glycolytic route and tricarboxylic acid cycle (14CO2 yield from carbons 3, 4, and 6 of glucose) increased during development. The flux through the glutamate-γ-aminobutyric route (14CO2 yield from carbon 2-carbon 6 of glucose) also showed an increase with development. In contrast, the proportion of glucose metabolized via the pentose phosphate pathway was markedly decreased as development progressed. The artificial electron acceptor, phenazine methosulfate, was used as a probe to investigate the effect of alterations in the redox state of NADP + NADPH couple on a number of NADP-linked systems in developing brain. Phenazine methosulfate produced a massive (20- to 50-fold) stimulation of the pentose phosphate pathway, in contrast, the incorporation of glucose carbon into fatty acids and flux through the glutamate-γ-aminobutyrate shunt were sharply decreased. The effects of phenazine methosulfate on the incorporation of glucose into glyceride glycerol, on the flux of glucose through the pyruvate dehydrogenase reaction and tricarboxylic acid cycle, all processes linked to the NAD + NADH couple, appeared to be minimal in the brain at the stages of development studied, i.e., 1, 5, 10, 20 days, and in the adult rat. The significance of the massive reserve potential of the pentose phosphate pathway in the developing brain is discussed.

Metabolic and molecular action of Trigonella foenum-graecum (fenugreek) and trace metals in experimental diabetic tissues

Diabetes mellitus is a heterogeneous metabolic disorder characterized by hyperglycaemia resulting in defective insulin secretion, resistance to insulin action or both. The use of biguanides, sulphonylurea and other drugs are valuable in the treatment of diabetes mellitus; their use, however, is restricted by their limited action, pharmaco-kinetic properties, secondary failure rates and side effects. Trigonella foenum-graecum, commonly known as fenugreek, is a plant that has been extensively used as a source of antidiabetic compounds from its seeds and leaf extracts. Preliminary human trials and animal experiments suggest possible hypoglycaemic and anti-hyperlipedemic properties of fenugreek seed powder taken orally. Our results show that the action of fenugreek in lowering blood glucose levels is almost comparable to the effect of insulin. Combination with trace metal showed that vanadium had additive effects and manganese had additive effects with insulin on in vitro system in control and diabetic animals of young and old ages using adipose tissue. The Trigonella and vanadium effects were studied in a number of tissues including liver, kidney, brain peripheral nerve, heart, red blood cells and skeletal muscle. Addition of Trigonella to vanadium significantly removed the toxicity of vanadium when used to reduce blood glucose levels. Administration of the various combinations of the antidiabetic compounds to diabetic animals was found to reverse most of the diabetic effects studied at physiological, biochemical, histochemical and molecular levels. Results of the key enzymes of metabolic pathways have been summarized together with glucose transporter, Glut-4 and insulin levels. Our findings illustrate and elucidate the antidiabetic/insulin mimetic effects of Trigonella, manganese and vanadium.

Effect of Aldose Reductase Inhibitor (Sorbinil) on Integration of Polyol Pathway, Pentose Phosphate Pathway, and Glycolytic Route in Diabetic Rat Lens

This study examines the effect of an aldose reductase inhibitor (sorbinil) on the flux of specifically labeled glucose through alternative pathways of metabolism in the lens of normal and diabetic rats 1 wk after the induction of diabetes with alloxan. In the diabetic rat lens, there was an apparent increase in the flux of glucose through the pentose phosphate pathway (PPP), as measured by the difference in the yields of 14CO2 from]1-14C[glucose and [6-14C]glucose [C1–C6], this value was 0.087 ± 0.005 and 0.263 ± 0.034 μmol · g lens−1 · h (mean + SE of 6 values) for control and diabetic rats, respectively; sorbinil treatment decreased the values to 0.065 ± 0.008 and 0.171 ± 0.028, respectively. With glucose tritiated on carbon 2 or 3, it has been shown that the flux of glucose through the polyol route is increased, whereas the flux through the glycolytic pathway is decreased in the diabetic rat lens; both are restored toward normal in the sorbinil-treated diabetic group. These results suggest that the dual effects of diabetes in increasing 1) the lens content of glucose and glucose 6-phosphate and 2) the flux of glucose in the polyol pathway will result in an increased utilization of NADPH and production of NADH, factors favoring the flow of glucose through the PPP and restricting the glycolytic route in the diabetic rat lens. The inhibition of aldose reductase by sorbinil tends to normalize the redox state of the nicotinamide nucleotides, reimposing the NADPH limitation on the PPP and increasing the availability of NAD+ for the glycolytic route.

Renal Hypertrophy in Experimental Diabetes: Changes in Pentose Phosphate Pathway Activity

An examination was made of the effect of different periods of experimental diabetes on the activity of the pentose phosphate pathway in rat kidney. A rapid increase in kidney weight, expressed both in absolute terms and in terms of body weight, occurred shortly after the induction of diabetes. The activity of the enzymes of the oxidative segment of the pentose phosphate pathway and the flux of glucose through the pathway were both increased during the first 7 days after induction of diabetes. Thereafter, enzyme activity returned toward control levels, but the increased functional activity of the pathway, as measured using specifically labeled glucose, persisted. In contrast, transketolase was significantly depressed at the time of most rapid kidney growth. A positive correlation was found between the rate of kidney growth and the change in activity of glucose-6-phosphate dehydrogenase and a negative correlation with changes in transketolase activity. The possible roles of the oxidative and nonoxidative segments of the pentose phosphate pathway in the kidney in early diabetes-induced renal hypertrophy are discussed.

Evidence for the existence and functional activity of the pentose phosphate pathway in the large particle fraction isolated from rat tissues.

Abstract Evidence is presented to show that all the enzymes of the pentose phosphate pathway are present in the 12, 000 g particulate fraction in a wide range of tissues. That these are constituent and functional is suggested by the findings that: (1) significant activity of the entire sequence of enzymes could be revealed after treatment of the washed particulate fraction by freezing and thawing and Triton X-100 or by mechanical disruption in phosphate buffer although no such activity could be detected in untreated particles; (2) the relative rates of 14 CO 2 production from glucose labelled on C-1, C-2 or C-6 were consistent with the operation of the cycle; (3) accumulation of intermediates of the pathway occurred when the liver large particle fraction was incubated in a fortified medium. The tissues studied were rat liver, ethionine-induced hepatoma, brain, adrenal gland, ovary and uterus.

Carbohydrate metabolism of mammary tissue III. Factors in the regulation of pathways of glucose catabolism in the mammary gland of the rat

Abstract 1. 1. The effect of insulin in stimulating the formation of 14 CO 2 from [ I - 14 C]glucose by mammary gland slices has been further investigated. 2. 2. No changes were found in the activities of hexokinase, G-6-P or 6-PG dehydrogenases, TPNH-cytochrome c reductase or TPNH-glutathione reductas ein the mammary gland following insulin treatment. 3. 3. Insulin has a much smaller effect on the oxidation of carbon- I of glucose and the incorporation of glucose carbon into fatty acids by mammary tissue from pregnant rats than on mammary tissue from lactating rats. 4. 4. Phenazine methosulphate, pyocyanin and methylene blue all cause a preferential stimulation of the oxidation of carbon- I of glucose; DNP and dicoumarol increase the oxidation of carbon-6 of glucose by mammary gland slices. 5. 5. High glucose concentrations increased the oxidation of carbon- I of glucose and the incorporation of glucose carbon into fatty acids; the oxidation of carbon-6 of glucose appeared to be independent of the glucose concentration. 6. 6. The action of insulin and of redox systems on the metabolism of glucose have been discussed; it is suggested that the supply of oxidised TPN is one of the factors controlling the activity of the pentose phosphate pathway.