A. Leslie Greenbaum

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.

Alternative pathways of glucose utilization in brain; changes in the pattern of glucose utilization in brain resulting from treatment of rats with 6-aminonicotinamide.

Abstract: The effect of 6‐aminonicotinamide (6AN) treatment on the activities of alternative pathways of glucose metabolism in 20‐day‐old rat brain was evaluated by measurements of yields of 14CO2 from glucose labeled with 14C on carbons 1, 2, 3 + 4, or 6 and uniformly labeled glucose, and from the incorporation of 14C from specifically labeled glucose into lipids by brain slices from cerebral hemispheres and cerebellum. At the highest dose of 6AN used (35 mg/kg body weight) there was a significant decrease in the 14CO2 yields via the pentose phosphate pathway, the glycolytic route, tricarboxylic acid (TCA) cycle, and via the glutamate‐γ‐aminobutyric acid pathway. Giving a graded series of doses (20–35 mg 6AN/kg body weight) revealed a hierarchy of responses in which the pentose phosphate pathway, lactate, glyceride‐glycerol, and fatty acid formation were most sensitive, followed, in sequence, by the pyruvate dehydrogenase reaction, the glutamate‐γ‐aminobutyrate route and, finally, the TCA cycle. The nature of the blocks in the various pathways was examined by the use of metabolite profiles.

Age-Related Changes in Enzymes of Rat Brain

The flux of glucose in the pathways of acetyl group formation and disposal and the activities of a range of enzymes related to these processes have been measured in the brains of rats aged between 1 day and 24 months. The pattern of enzyme change is such that those systems involved in hydrogen transfer appear to increase disproportionately, relative to the glycolytic flux in the aged brain. It is suggested that these increases are an essential corollary to the need for a maintained glycolytic flux in a tissue dependent upon glucose as a fuel and one in which alternative routes of NADH oxidation diminish with age.

Alternative Pathways of Glucose Utilization in Brain: Changes in the Pattern of Glucose Utilization and of the Response of the Pentose Phosphate Pathway to 5‐Hydroxytryptamine During Aging

Abstract: The oxidation of differentially labelled glucose, pyruvate and glutamate in brain slices from rats aged 20 days to 26 months has been studied and the partition of the glucose used into the glycolytic‐tricarboxylic acid cycle pathway, the pentose phosphate pathway and the glutamate‐GABA shunt has been calculated. Over the time range 4 to 26 months, there is an approximately 20% decrease in the production of CO2 via the glycolytic‐tricarboxylic acid cycle route, as there is in the rate of glucose phosphorylation. The glutamate‐GABA pathway falls by about 50% over this same time span. The broad activity of the pentose phosphate pathway falls rapidly and cannot be detected in the brains of rats aged 18 months or more, whereas the fully stimulated pathway, i.e. in the presence of the artificial electron acceptor phen‐azine methosulphate, declines only marginally over this period, falling sharply only after 23 months. The pentose phosphate pathway is stimulated by the presence of 5‐hy‐droxytryptamine and this stimulation appears to increase with age.

Reciprocal Control of Pyruvate Dehydrogenase Kinase and Phosphatase by Inositol Phosphoglycans DYNAMIC STATE SET BY “PUSH-PULL” SYSTEM

Reversible phosphorylation of proteins regulates numerous aspects of cell function, and abnormal phosphorylation is causal in many diseases. Pyruvate dehydrogenase complex (PDC) is central to the regulation of glucose homeostasis. PDC exists in a dynamic equilibrium between de-phospho-(active) and phosphorylated (inactive) forms controlled by pyruvate dehydrogenase phosphatases (PDP1,2) and pyruvate dehydrogenase kinases (PDK1–4). In contrast to the reciprocal regulation of the phospho-/de-phospho cycle of PDC and at the level of expression of the isoforms of PDK and PDP regulated by hormones and diet, there is scant evidence for regulatory factors acting in vivo as reciprocal “on-off” switches. Here we show that the putative insulin mediator inositol phosphoglycan P-type (IPG-P) has a sigmoidal inhibitory action on PDK in addition to its known linear stimulation of PDP. Thus, at critical levels of IPG-P, this sigmoidal/linear model markedly enhances the switchover from the inactive to the active form of PDC, a “push-pull” system that, combined with the developmental and hormonal control of IPG-P, indicates their powerful regulatory function. The release of IPGs from cell membranes by insulin is significant in relation to diabetes. The chelation of IPGs with Mn2+ and Zn2+ suggests a role as “catalytic chelators” coordinating the traffic of metal ions in cells. Synthetic inositol hexosamine analogues are shown here to have a similar linear/sigmoidal reciprocal action on PDC exerting push-pull effects, suggesting their potential for treatment of metabolic disorders, including diabetes.

The insulin-mimetic action of Mn2+: Involvement of cyclic nucleotides and insulin in the regulation of hepatic hexokinase and glucokinase

Manganese causes a significant rise in hepatic glucokinase and hexokinase in 16-day-old suckling rats, and has an insulinomimetic effect in producing a precocious emergence of glucokinase (EC 2.7.1.2) and a rise in the low Km, hexokinases (EC 2.7.1.1) activities. These enzyme changes occur within 6 hr of manganese administration and there are accompanying increases in plasma insulin and hepatic cyclic GMP. That the effect of manganese is at a site other than, or in addition to, insulin secretion is suggested by the significant increases in glucokinase and hexokinase in 16-day-old streptozotocin-diabetic rats; in this group there is also an increase in hepatic cGMP similar in time scale to that of the normal-manganese-treated group. The effects of manganese and insulin were not additive. It is proposed that one site of action of manganese may be at the level of cyclic GMP systems. The results are also discussed in relation to the known action of manganese at the level of the protein phosphatases.

Pyrimidine nucleotide synthesis in the rat kidney in early diabetes

Early renal hypertrophy of diabetes is associated with increases in the tissue content of RNA, DNA, and sugar nucleotides involved in the formation of carbohydrate-containing macromolecules. We have previously reported an increase in the activity of enzymes of the de novo and salvage pathways of purine synthesis in early diabetes; the present communication explores the changes in the pathways of pyrimidine synthesis. Measurements have been made of key enzymes of the de novo and salvage pathways at 3, 5, and 14 days after induction of diabetes with streptozotocin (STZ), phosphoribosyl pyrophosphate (PPRibP), and some purine and pyrimidine bases. Carbamoyl-phosphate synthetase II, the rate-limiting enzyme of the de novo route, did not increase in the first 5 days after STZ treatment, the period of most rapid renal growth; a significant rise was seen at 14 days (+38%). Dihydroorotate dehydrogenase, a mitochondrial enzyme, showed the most marked rise (+147%) at 14 days. The conversion of orotate to UMP, catalyzed by the enzymes of complex II, was increased at 3 days (+42%), a rise sustained to 14 days. The salvage route enzyme, uracil phosphoribosyltransferase (UPRTase), showed a pattern of change similar to complex II. The effect of the decreased concentration of PPRibP on the activities of CPSII, for which it is an allosteric activator, and on activities of OPRTase and UPRTase, for which it is an essential substrate, is discussed with respect to the relative Ka and Km values for PPRibP and the possibility of metabolite channeling.

Effects of long-term experimental diabetes on adrenal gland growth and phosphoribosyl pyrophosphate formation in growth hormone-deficient dwarf rats

The availability of growth hormone (GH)‐deficient dwarf rats with otherwise normal pituitary function provides a powerful tool to examine the relative role of hyperglycaemia and the reordering of hormonal factors in the hypertrophy‐hyperfunction of the adrenal gland that is seen in experimental diabetes. Here, we examine the effects of long‐term (6 months) experimental diabetes on the growth of the adrenal glands; their content of phosphoribosyl pyrophosphate (PRPP); and the activity of the PRPP synthetase, G6P dehydrogenase and 6PG dehydrogenase enzymes in GH‐deficient dwarf rats compared to heterozygous controls. These parameters were selected in view of the known role of PRPP in both de novo and salvage pathways of purine and pyrimidine synthesis and in the formation of NAD, and in view of the role of the oxidative enzymes of the pentose phosphate pathway in both R5P formation and the generation of the NADPH that is required in reductive synthetic reactions. This study shows that GH deficiency prevents the increase in adrenal gland weight, PRPP synthetase, PRPP content and G6P dehydrogenase and 6PG dehydrogenase. This contrasts sharply with the heterozygous group that showed the expected increase in these parameters. The blood glucose levels of the groups of long‐term diabetic rats, both GH‐deficient and heterozygous, remained at an elevated level throughout the experiment. These results are fully in accord with earlier evidence from studies with somatostatin analogues which showed that the GH‐insulin‐like growth factor I (IGF‐I)‐axis plays a key role in the adrenal diabetic hypertrophy‐hyperfunction syndrome.

Effects of long-acting somatostatin analogues on adrenal growth and phosphoribosyl pyrophosphate formation in experimental diabetes

Adrenal growth and increased adrenal function occur in experimental diabetes. Previously, we have shown that phosphoribosyl pyrophosphate (PRPP) and PRPP synthetase increased rapidly between 3 and 7 days after induction of diabetes with streptozotocin (STZ), with less marked changes in enzymes of the pentose phosphate pathway. The present study examines the earlier phase of 1–3 days following induction of diabetes, seeking to elucidate whether control of PRPP production is a result of diabetic hyperglycaemia, or to a more general re‐ordering of hormonal factors. To investigate this question, the role of insulin and two different long‐acting somatostatin analogues, Angiopeptin and Sandostatin, were used in a well‐established animal model. PRPP was chosen specifically as a target for these studies in view of its central role in nucleotide formation and nicotinamide mononucleotide synthesis via Nampt which is the rate‐limiting step in the synthesis of NAD and which has been shown to have multiple roles in cell signalling in addition to its known function in glycolysis and energy production. Treatment with the somatostatin analogues ab initio effectively abolished the adrenal growth, the increase in PRPP formation and the rise of PRPP synthetase activity in the first 7 days of diabetes, without having any significant effect on blood glucose values. This suggests that elevated glucose per se is not responsible for the diabetic adrenal hypertrophy and implies that growth factors/hormones, regulated by somatostatin analogues, play a significant role in adrenal growth processes.

Uridine and cytidine nucleotide synthesis in renal hypertrophy : biochemical differences in response to the growth stimulus of diabetes and unilateral nephrectomy

The effects of unilateral nephrectomy (UN) and streptozotocin (STZ) diabetes on the activities of enzymes involved in uridine and cytidine synthesis in early renal growth (3-14 days after stimulus to growth) have been compared. Measurements were also made of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) and of glucose 6-phosphate (G6P), UDP-glucose, and glycogen, in relation to phosphoribosyl pyrophosphate, ribonucleotide, and complex carbohydrate formation. There were striking differences in the activities of CTP synthetase, G6PDH, and 6PGDH in the two conditions, with a three-fold increase in all three enzymes at 3 and 5 days and a two-fold increase above basal values at 14 days of STZ diabetes. The UN group showed no significant change in CTP synthetase at any stage and the activity of G6PDH and 6PGDH only kept pace with renal growth. Changes in routes of uridine synthesis were less marked, with a more rapid rise in carbamoyl-phosphate synthetase (glutamine) and a lesser response of dihydroorotate dehydrogenase in the UN relative to the STZ-diabetic groups. The enzymes of complex II and of uracil phosphoribosyltransferase showed essentially similar patterns during renal hypertrophy in UN and STZ diabetes. The parallel increase in CTP synthetase, G6PDH, and 6PGDH in the kidney in diabetes, also known to increase in growth situations in hepatomas and in renal tumors, is discussed in relation to hormone signals involved in renal growth. The importance of the concentration of CTP, and thus of CTP synthetase, in the CTP-cytidyltransferase reaction, an enzyme with a high Km for CTP, makes the present observation of the striking increase in CTP synthetase in STZ diabetes of particular interest in relation to phosphatidylcholine formation and hormone signal transduction.