Changes of glucose metabolism and skin-collagen neogenesis in vitamin B6 deficiency

The mechanism of pellagrous changes in skin caused by a deficiency of vitamin B6 was studied in respect to neogenesis of proline in skin collagen and glucose metabolism. In vitamin B6 deficiency the insulin/glucagon coefficient in serum decreased significantly from 3.02 to 2.32, indicating a metabolic change towards gluconeogenesis. A deficiency of vitamin B6 caused a decrease in the levels of vitamin B6-dependent enzymes, such as ornithine aminotransferase, alanine aminotransferase, and aspartate aminotransferase, which also contribute to gluconeogenesis. Because the conversion of ornithine to proline via pyrroline-5-carboxylate was suppressed due to the decrease in ornithine aminotransferase activity, the amount of proline in the skin collagen fraction also decreased significantly in vitamin B6-deficient rats compared with the pair-fed control. These results suggest that the pellagrous lesions in vitamin B6-deficiency are caused by an impaired synthesis of proline from ornithine, which results in the suppression of collagen neogenesis in the skin.     

Experimental diabetes causes mitochondrial loss and cytoplasmic enrichment of pyridoxal phosphate and aspartate aminotransferase activity

The streptozotocin diabetic rat was selected as a model to study how insulin deficiency alters vitamin B6 utilization by focusing on pyridoxal phosphate levels and aspartate aminotransferase activities in liver tissues. Diabetes of 15 weeks' duration lowered plasma pyridoxal phosphate levels by 84%. Normal plasma pyridoxal phosphate was 480 pmole/ml. Fractionation of liver into mitochondrial and extramitochondrial compartments demonstrated that diabetes caused a 43% diminution in mitochondrial pyridoxal phosphate per gram of liver. There was no cytoplasmic change in these diabetic rats. Mitochondrial aspartate aminotransferase activity was decreased 53% per gram of diabetic liver and cytoplasmic aspartate aminotransferase activity was elevated 3.4-fold. Damage to diabetic mitochondria during preparation procedures could not account for the rise in cytoplasmic aspartate aminotransferase activity. Electrophoresis showed that in the diabetic cytoplasm both cathodal and anodal forms of the enzyme were elevated. Speculations concerning mitochondrial loss and cytoplasmic gain of enzyme activity as well as those on the reduction of plasma pyridoxal phosphate in the diabetic rat are presented.     

Glycogen and glycogen enzymes in the liver and striated muscle of rats under altered thyroid states

Changes induced in liver and striated muscle glycogen and glycogen enzymes (glycogen synthetase, glycogen phosphorylase and alpha-amylase) by hypothyroidism and hyperthyroidism in rats have been determined. There were no changes in liver glycogen synthetase, phosphorylase and amylase activities in the hypothyroid group. Hyperthyroid rats showed lower liver glycogen synthetase, phosphorylase a and amylase activities. In muscle, hypothyroid rats had lower phosphorylase activity. In the hyperthyroid group glycogen synthetase was increased.--The results presented do not completely agree with the glycogen levels found in both tissues studied, and they are obviously more related to other factors such as glucose availability. It can be concluded that under the conditions studied, the glycogen enzyme levels could not alone explain the variations of glycogen levels.     

The effect of long-term enrichment of diet with selenium, vitamin E and B15 on the activity of certain enzymes in rat brain

The aim of this study was tracing of changes in the activity of glutathione peroxidase (GSHPx), glutathione transferase (GSH S-Tr), aspartate aminotransferase (AspAT) and alanine aminotransferase (A1AT) in the brain as a result of diet enrichment with antioxidants: selenium (Se), vitamin E and vitamin B15 (pangamic acid). The experiment was carried out on Wistar rats with initial body weight 150 g. Following prolonged enrichment of diet with Se (0.1 ppm of sodium selenite), vitamin E (6 mg/100 g of diet) and vitamin B15 (2.5 mg/100 g of diet) the following results were obtained. The activity of GSHPx in brain microsomes was not changed after one year of vitamin E administration when it was measured against hydrogen hydroxide and against cumene hydrochloride; vitamin E administration increased the activity of GSH S-Tr in the cytoplasmic fraction of brain cells. Diet enrichment with selenium increased after 12 and 18 months the activity of GSHPx measured against both substrates, and GSH S-Tr activity increased considerably. Presence of vitamin B15 in diet reduced GSHPx activity after one-year or longer administration, after 18 months the activity of GSH S-Tr was reduced also. No changes were noted in the activity of AspAT and A1AT.     

Role of AMP on the activation of glycogen synthase and phosphorylase by adenosine, fructose, and glutamine in rat hepatocytes

The mechanism for glycogen synthesis stimulation produced by adenosine, fructose, and glutamine has been investigated. We have analyzed the relationship between adenine nucleotides and glycogen metabolism rate-limiting enzymes upon hepatocyte incubation with these three compounds. In isolated hepatocytes, inhibition of AMP deaminase with erythro-9-(2-hydroxyl-3nonyl)adenine further increases the accumulation of AMP and the activation of glycogen synthase and phosphorylase by fructose. This ketose does not increase cyclic AMP or the activity of cyclic AMP-dependent protein kinase. Adenosine raises AMP and ATP concentration. This nucleotide also activates glycogen synthase and phosphorylase by covalent modification. The correlation coefficient between AMP and glycogen synthase activity is 0.974. Nitrobenzylthioinosine, a transport inhibitor of adenosine, blocks (by 50%) the effect of the nucleoside on AMP formation and glycogen synthase but not on phosphorylase. 2-Chloroadenosine and N6-phenylisopropyladenosine, nonmetabolizable analogues of adenosine, activate phosphorylase (6-fold) without increasing the concentration of adenine nucleotides or the activity of glycogen synthase. Cyclic AMP is not increased by adenosine in hepatocytes from starved rats but is in cells from fed animals. [Ethylenebis (oxyethylenenitrilo)]tetraacetic acid (EGTA) blocks by 60% the activation of phosphorylase by adenosine but not that of glycogen synthase. Glutamine also increases AMP concentration and glycogen synthase and phosphorylase activities, and these effects are blocked by 6-mercaptopurine, a purine synthesis inhibitor. Neither adenosine nor glutamine increases glucose 6-phosphate. It is proposed that the observed efficient glycogen synthesis from fructose, adenosine, and glutamine is due to the generation of AMP that activates glycogen synthase probably through increases in synthase phosphatase activity. It is also concluded that the activation of phosphorylase by the above-mentioned compounds can be triggered by metabolic changes.     

Phosphorylase activity and glycogen content in cerebral and myocardium tissues in rats of different age

The phosphorylase activity (EC 2.4.1.1) and the glycogen content were determined in the brain and myocardium of adult and old rats. In the cortex and while substance of great cerebral hemispheres, the phosphorylase a activity decreases and the phosphorylase b activity increases, while the total enzyme activity remains unchanged with aging. In the myocardium, the activity of phosphorylase a increases, while that of phophorylase b decreases against a background of an insignificant decrease in the total phosphorylase activity. During aging, the glycogen content decreases sharply in the myocardium, whereas in the cerebral tissue it remains unchanged. Age peculiarities of the adrenaline effect upon myocardial phosphorylase activity are presented.     

Effects of diabetes and glycogen on the distribution between soluble and particulate fractions of activities of enzymes involved in hepatic glycogen metabolism.

The large decreases in hepatic glycogen associated with alloxan diabetes in fed rats were accompanied by apparent decreases in total activities of glycogen synthase, phosphorylase, protein kinase and synthase phosphatase determined on 8000 × g supernatants of liver homogenates. Inclusion of 4% glycogen in the extraction buffer normalized total soluble activities of synthase in the diabetic. Whereas inclusion of 4% glycogen in the extraction buffer doubled total soluble phosphorylase, total activity remained lower in the diabetic than in the normal. Extraction and assay of soluble protein kinase were unaffected by added glycogen. When activities were determined on whole homogenates, total glycogen synthase activities were the same in normal and diabetic liver. Although the decreases in total activities of phosphorylase, kinase and phosphatase were less when determined on whole homogenates of livers from diabetic rats, the diabetes-related decreases in total activities remained significant. Therefore, it appears that while alloxan diabetes results in absolute decreases in total hepatic activities of phosphorylase, kinase and phosphatase, it may also result in redistribution of hepatic synthase and phosphorylase between soluble and particulate fractions, a phenomenon possibly related to tissue glycogen concentrations. Such a redistribution might be involved in the lack of control of hepatic glycogenesis observed in alloxan diabetic rats.     

Glycogen synthase activation by sugars in isolated hepatocytes

We have investigated the activation by sugars of glycogen synthase in relation to (i) phosphorylase a activity and (ii) changes in the intracellular concentration of glucose 6-phosphate and adenine nucleotides. All the sugars tested in this work present the common denominator of activating glycogen synthase. On the other hand, phosphorylase a activity is decreased by mannose and glucose, unchanged by galactose and xylitol, and increased by tagatose, glyceraldehyde, and fructose. Dihydroxyacetone exerts a biphasic effect on phosphorylase. These findings provide additional evidence proving that glycogen synthase can be activated regardless of the levels of phosphorylase a, clearly establishing that a nonsequential mechanism for the activation of glycogen synthase occurs in liver cells. The glycogen synthase activation state is related to the concentrations of glucose 6-phosphate and adenine nucleotides. In this respect, tagatose, glyceraldehyde, and fructose deplete ATP and increase AMP contents, whereas glucose, mannose, galactose, xylitol, and dihydroxyacetone do not alter the concentration of these nucleotides. In addition, all these sugars, except glyceraldehyde, increase the intracellular content of glucose 6-phosphate. The activation of glycogen synthase by sugars is reflected in decreases on both kinetic constants of the enzyme, M0.5 (for glucose 6-phosphate) and S0.5 (for UDP-glucose). We propose that hepatocyte glycogen synthase is activated by monosaccharides by a mechanism triggered by changes in glucose 6-phosphate and adenine nucleotide concentrations which have been described to modify glycogen synthase phosphatase activity. This mechanism represents a metabolite control of the sugar-induced activation of hepatocyte glycogen synthase.     

Aminotransferase activity in the brain of rats of various ages during hypothermia

Studies have been made on the activity of aspartate and alanine aminotransferase in the brain of 1, 4, 12-14, 16, 22 days, 1, 1 1/2, 3 months and 2 years old rats under hypothermic conditions (20-19 degrees C). It was shown that hypothermia decreases both total and specific activities of the enzymes in the developing brain. Alanine aminotransferase activity in brain homogenates determined at 37 and 20-19 degrees C, exhibits more significant changes than of aspartate aminotransferase.     

Changes in the activity of enzymes,participating in glycogen metabolism of alloxan diabetic rats

Summary Alloxan diabetes induced in white rats by intraperitoneal injection of Aloxan-monohydrate (15 mg/100 g body weight) was used to study changes in the glycogen phosphorylase a and b, phosphoprotein phosphatases and hexokinase activities under insulin deficiency conditions. Among the enzymes studied, an increase in muscle phosphorylase a activity as well as the a/b ratio have been obtained. In diabetic muscle phosphoprotein phosphatases and hexokinase activities were diminished.AMP increased the liver glycogen phosphorylase activity twice in diabetic rats whereas in normal animals the enzyme was less sensitive to this effector. The changes in liver hexokinase activity at diabetes were not connected and correlated with the altered phosphorylase and protein phosphatase activities.The logical chain of probable molecular events taking place in muscle glycogen metabolism under the conditions of insulin deficiency is offered.     

Hypermethioninemia provokes oxidative damage and histological changes in liver of rats

In the present study we evaluated the effect of chronic methionine administration on oxidative stress and biochemical parameters in liver and serum of rats, respectively. We also performed histological analysis in liver. Results showed that hypermethioninemia increased chemiluminescence, carbonyl content and glutathione peroxidase activity, decreased total antioxidant potential, as well as altered catalase activity. Hypermethioninemia increased synthesis and concentration of glycogen, besides histological studies showed morphological alterations and reduction in the glycogen/glycoprotein content in liver. Serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and glucose were increased in hypermethioninemic rats. These findings suggest that oxidative damage and histological changes caused by methionine may be related to the hepatic injury observed in hypermethioninemia.     

Intracellular predominance of the pyridoxal 5'-phosphate form of aspartate aminotransferase in Escherichia coli B and reversible transformation of this form by extracellular substances

The intracellular proportion of the pyridoxal 5'-phosphate form of aspartate aminotransferase to the total enzyme in E. coli B cells was determined by a newly devised method, dependent on selective inactivation of the intracellular pyridoxal 5'-phosphate form of the enzyme by extracellularly added sodium borohydride. A large portion (80-99%) of the intracellular aspartate aminotransferase was in pyridoxal 5'-phosphate form in both natural and synthetic medium-grown bacterial cells. The intracellular predominancy of pyridoxal 5'-phosphate did not vary during the growth of bacteria and during incubation of bacterial cells in various kinds of buffers with different pH values. In contrast, the saturation levels generally used to describe in vivo the proportions of the apo and holo vitamin B6-dependent enzymes did not reflect the intracellular amount of the pyridoxal 5'-phosphate (holo) form of aspartate aminotransferase probably because the intracellular pyridoxal 5'-phosphate form was changed to an apo form by the disruption of bacterial cells for preparing crude extract. Various extracellularly-added vitamin B6 antagonists decreased the intracellular amount of pyridoxal 5'-phosphate without decrease in the total intracellular activity of the enzyme. The modified forms were stable in E. coli B cells and reversed into pyridoxal 5'-phosphate form by incubation of the antagonist-treated cells in the buffer containing pyridoxal. The present results showed that the sodium borohydride reduction method can be used for further analysis of the in vivo interaction of pyridoxal 5'-phosphate and apoaspartate aminotransferase. The fact that about 50% of the intracellular pyridoxal 5'-phosphate form was changed to a modified form without impairment of cell growth in the presence of 4-deoxypyridoxine, and that about 50% of intracellular modified aspartate aminotransferase was reversed to pyridoxal 5'-phosphate by the removal of antagonist followed by incubation suggested that there exists characteristically 2 different fractions of pyridoxal 5'-phosphate forms of aspartate aminotransferase in E. coli cells.     

Metabolic effects of propionate in normal and vitamin B12-deficient rats

1. Administration of propionate caused a twofold increase in the concentrations of lactate and pyruvate in the blood of vitamin B(12)-deficient rats, whereas there was a slight decrease in lactate and a 50% increase in pyruvate in normal rats. 2. Concentrations of total ketone bodies in the blood of normal rats were not significantly altered by propionate administration but the [3-hydroxybutyrate]/[acetoacetate] ratio decreased from 3.0 to 2.0. In the vitamin B(12)-deficient rats there was a 40% decrease in total ketone bodies and a change in the ratio from 3.4 to 1.2. 3. The changes in the concentration of ketone bodies in freeze-clamped liver preparations were similar in pattern to those observed in blood. 4. Propionate administration caused a decrease in the concentration of acetyl-CoA in the livers of both groups of animals, but the absolute decrease was greater in the vitamin B(12)-deficient group. The decrease in the concentration of CoA was similar in both groups. 5. As in blood, there were threefold increases in the concentrations of lactate and pyruvate in the livers of the vitamin B(12)-deficient rats after propionate administration, whereas there was no significant change in the concentrations of these metabolites in the normal rats. 6. There was a 50% inhibition of glucose synthesis in perfused livers from vitamin B(12)-deficient rats when lactate and propionate were substrates as compared with lactate alone. 7. It is concluded that the conversion of lactate into glucose is inhibited in vitamin B(12)-deficient rats after propionate administration, and that this effect is due to inhibition of the pyruvate carboxylase step resulting from a decrease in acetyl-CoA concentration and a postulated increase in methylmalonyl-CoA concentration.     

Inhibition of muscle glycogen phosphorylase b by vitamin B2 and its coenzyme forms

Kinetic studies have demonstrated that vitamin B2 and its coenzyme forms FMN and FAD are potent inhibitors of glycogen phosphorylase b from rabbit skeletal muscle. The inhibition of the enzyme by flavins has a co-operative character (Hill coefficients exceed unity). Glycogen phosphorylase b bound to FMN or FAD does not reveal catalytic activity, whereas the enzyme bound to riboflavin retains about 16% of the initial catalytic activity.     

Interaction between glycogen and glycogen phosphorylase of Tetrahymena

The glycogen phosphorylase of Tetrahymena pyriformis complexes with glycogen as judged by its elution pattern from columns of Sepharose 6B. Complex formation does not occur with starch, amylose, or amylopectin, and neither do these polyglucans serve as primers for the enzyme. To study the association between the phosphorylase and glycogen particles in situ, Tetrahymena were grown under differing physiological conditions, phosphorylase was isolated and chromatographed on a Sepharose 6B column. Phosphorylase activity isolated from cells grown in the absence of glucose was only partially associated with glycogen, while in cells exposed to glucose for 30 min or more all the phosphorylase activity was associated with glycogen. The effects of culture age and anaerobiosis on the relative amounts of free and glycogen-bound enzyme in the cells were also studied. It was concluded from the in vivo experiments that there was no simple relation between the fraction of enzyme bound to glycogen and between cell glycogen content.     

Lithium's effects on rat liver glucose metabolism in vivo

Oral administration of lithium carbonate to fed-healthy rats strongly decreased liver glycogen content, despite the simultaneous activation of glycogen synthase and the inactivation of glycogen phosphorylase. The effect seemed to be related to a decrease in glucose 6-phosphate concentration and to a decrease in glucokinase activity. Moreover, in these animals lithium markedly decreased liver fructose 2,6-bisphosphate, which could be a consequence of the fall in glucose 6-phosphate and of the inactivation of 6-phosphofructo-2-kinase. Liver pyruvate kinase activity and blood insulin also decreased after lithium administration. Lower doses of lithium carbonate had less intense effects. Lithium administration to starved-healthy and fed-streptozotocin-diabetic rats caused a slight increase in blood insulin, which was simultaneous with increases in liver glycogen, glucose 6-phosphate, and fructose 2, 6-phosphate. Glucokinase, 6-phosphofructo-2-kinase, and pyruvate kinase activities also increased after lithium administration in starved-healthy and fed-diabetic rats. Lithium treatment activated glycogen synthase and inactivated glycogen phosphorylase in a manner similar to that observed in fed-healthy rats. Glycemia was not modified in any group of animals. These results indicate that lithium acts on liver glycogen metabolism in vivo in at least two different ways: one related to changes in insulinemia, and the other related to the direct action of lithium on the activity of some key enzymes of liver glucose metabolism.     

Effect of propionate pathway metabolites on the oxidative activity of liver mitochondria under normal conditions and in vitamin B12 deficiency]

We performed a comparative investigation in vivo and in vitro of the propionate and methylmalonate effect on oxidative activity of liver mitochondria in control and vitamin B12-deficient rats and found that efficiency of the effects were less pronounced in vitamin B12-deficient rats. It is also shown that the rates of respiration and phosphorylation decreased in liver mitochondria of vitamin B12-deficient rats.     

Development of oxidative stress and cell damage in the liver of rats with water-immersion restraint stress

We examined how oxidative stress and cell damage develop in the liver of rats subjected to water-immersion stress (WIRS). In rats subjected to WIRS for 1.5, 3 or 6 h, serum alanine aminotransferase and aspartate aminotransferase activities increased time-dependently. In the liver tissue, vacuolization and apoptosis occurred at 1.5 h of WIRS and vacuolization further developed without further appearance of apoptosis at 3 h or 6 h. Serum lipid peroxide (LPO) and NOx (nitrite/nitrate) concentrations increased at 3 h of WIRS and these increases were enhanced at 6 h. In liver tissue, increases in LPO and NOx concentrations and myeloperoxidase activity and decreases in ascorbic acid and reduced glutathione concentrations and superoxide dismutase activity occurred at 3 h of WIRS and these changes were enhanced at 6 h, although vitamin E concentration and xanthine oxidase activity were unchanged. These results indicate that oxidative stress in the liver of rats with WIRS develops after the appearance of cell damage in the tissue, and suggests that oxidative stress is caused through disruption of the antioxidant defense system and increases in NO generation and neutrophil infiltration in the liver, which may contribute to the progression of cell damage in the tissue.     

Observations on the histochemically demonstrable liver glycogen phosphorylase activity and native glycogen under different nutritional conditions

Summary In this study a histochemical demonstration of glycogen phosphorylase activity and native glycogen in the livers from normally fed, overfed and starved rats was performed.It was found that the amount and localization of phosphorylase activity well corresponded to the amount and localization of the native glycogen. A change of the glycogen content in the liver also resulted in a change of the histochemically demonstrable liver glycogen phosphorylase activity.It is concluded that the presence of tissue bound glycogen and undissolved glycogenphosphorylase complexes are necessary for positive histochemical demonstration of liver glycogen phosphorylase activity.This work was supported by a grant from the Finnish Veterinary Medical Foundation.     

Studies on the alpha-adrenergic activation of hepatic glucose output. I. Studies on the alpha-adrenergic activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase in isolated rat liver parenchymal cells.

Epinephrine and the alpha-adrenergic agonist phenylephrine activated phosphorylase, glycogenolysis, and gluconeogenesis from lactate in a dose-dependent manner in isolated rat liver parenchymal cells. The half-maximally active dose of epinephrine was 10-7 M and of phenylephrine was 10(-6) M. These effects were blocked by alpha-adrenergic antagonists including phenoxybenzamine, but were largely unaffected by beta-adrenergic antagonists including propranolol. Epinephrine caused a transient 2-fold elevation of adenosine 3':5'-monophosphate (cAMP) which was abolished by propranolol and other beta blockers, but was unaffected by phenoxybenzamine and other alpha blockers. Phenoxybenzamine and propranolol were shown to be specific for their respective adrenergic receptors and to not affect the actions of glucagon or exogenous cAMP. Neither epinephrine (10-7 M), phenylephrine (10-5 M), nor glucagon (10-7 M) inactivated glycogen synthase in liver cells from fed rats. When the glycogen synthase activity ratio (-glucose 6-phosphate/+ glucose 6-phosphate) was increased from 0.09 to 0.66 by preincubation of such cells with 40 mM glucose, these agents substantially inactivated the enzyme. Incubation of hepatocytes from fed rats resulted in glycogen depletion which was correlated with an increase in the glycogen synthase activity ratio and a decrease in phosphorylase alpha activity. In hepatocytes from fasted animals, the glycogen synthase activity ratio was 0.32 +/- 0.03, and epinephrine, glucagon, and phenylephrine were able to lower this significantly. The effects of epinephrine and phenylephrine on the enzyme were blocked by phenoxybenzamine, but were largely unaffected by propranolol. Maximal phosphorylase activation in hepatocytes from fasted rats incubated with 10(-5) M phenylephrine preceded the maximal inactivation of glycogen synthase. Addition of glucose rapidly reduced, in a dose-dependent manner, both basal and phenylephrine-elevated phosphorylase alpha activity in hepatocytes prepared from fasted rats. Glucose also increased the glycogen synthase activity ratio, but this effect lagged behind the change in phosphorylase. Phenylephrine (10-5 M) and glucagon (5 x 10(-10) M) decreased by one-half the fall in phosphoryalse alpha activity seen with 10 mM glucose and markedly suppressed the elevation of glycogen synthase activity. The following conclusions are drawn from these findings. (a) The effects of epinephrine and phenylephrine on carbohydrate metabolism in rat liver parenchymal cells are mediated predominantly by alpha-adrenergic receptors. (b) Stimulation of these receptors by epinephrine or phenylephrine results in activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase by mechanisms not involving an increase in cellular cAMP. (c) Activation of beta-adrenergic receptors by epinephrine leads to the accumulation of cAMP, but this is associated with minimal activation of phosphorylase or inactivation of glycogen synthase...