Anthony D. Morrison

Metabolic alterations in the human erythrocyte produced by increases in glucose concentration: The role of the polyol pathway

Human erythrocytes incubated in medium containing 50 mM glucose have increased intracellular sorbitol and fructose concentrations as compared with samples incubated with 5 mM glucose. Increased medium glucose concentration did not significantly alter total glucose consumption or lactate production. However, the intracellular lactate:pyruvate ratio rose, the concentrations of fructose diphosphate, and triose phosphates increased, and the 2,3-diphosphoglycerate concentration fell. [(14)C]O(2) production from glucose-1-(14)C also increased with increased medium glucose concentration. These changes are believed to reflect changes in the redox states of the diphosphopyridine nucleotide/reduced form of diphosphopyridine nucleotide (NAD/NADH) and nicotinamide-adenine dinucleotide phosphate/reduced form of nicotinamide-adenine dinucleotide phosphate (NADP/NADPH) couples resulting from increased activity of the polyol pathway. Addition of pyruvate to the incubation media prevented these changes. These studies illustrate that an increase in the red cells normal substrate, glucose, can produce changes in red cell metabolism.

Glucose utilization by the polyol pathway in human erythrocytes

Abstract Sorbitol is present in human erythrocytes in concentrations exceeding that in plasma, and is linearly related to the plasma glucose concentration. When erythrocytes are incubated in media containing increasing glucose concentrations, the intracellular sorbitol and fructose concentrations increase and free fructose appears in the media. At a medium glucose concentration of 5 mM approximately 3% of the glucose uptake is utilized for sorbitol and fructose synthesis.Glucose appears to be a physiological substrate for alditol:NADP oxidoreductase in the erythrocyte.

INCREASED CEREBROSPINAL-FLUID PRESSURE DURING TREATMENT OF DIABETIC KETOSIS

Abstract The cerebrospinal fluid (C.S.F.) pressure was continuously monitored in five patients during the first 10 hours of treatment for diabetic ketosis; in every case an abnormally high C.S.F. pressure developed after therapy had resulted in a rapid fall in plasma-glucose and a decrease in plasma osmolality. This increase in pressure was predicted by a hypothesis based upon previous studies of an experimental model for the development of acute cerebral œdema during treatment for diabetic ketosis. The development of increased C.S.F. pressure did not seem to be related to overhydration or to water intoxication, but may be related to the consequences of increased polyol pathway activity in brain. This tendency to develop abnormally high C.S.F. pressure during treatment for diabetic ketosis may provide an explanation for delayed recovery of consciousness, and for the development of fatal acute cerebral œdema.

Effects of Elevated Glucose Concentrations on the Metabolism of the Aortic Wall

The effects of elevated glucose concentrations on the metabolism of the aortic wall were examined in a preparation of tubular segments of rabbit descending thoracic aorta comprised of intima and media only. Increased medium glucose concentrations (20-50 mm) resulted in increased aortic sorbitol and fructose concentrations and an increased rate of fructose release into the medium. This increased flux through the polyol pathway can be explained as a consequence both of an increased free intracellular glucose concentration and of the kinetic characteristics of the alditol: NADP oxidoreductase and the l-iditol: NAD oxidoreductase isolated and partially purified from rabbit thoracic aorta. Incubation with elevated glucose concentrations for 2 or more hr was also associated with a significant increase in the water content of the tissue without a significant increase in the inulin space. The oxygen uptake of the tissues incubated with elevated glucose concentrations was significantly reduced; this appears to result from a limitation imposed by oxygen diffusion at physiological oxygen tensions. A compensatory increase in glycolysis and an increase in the aortic lactate/pyruvate concentration ratio were also observed. The oxygen uptake and lactate production of tissue incubated with 50 mm glucose could be preserved at rates observed in tissue incubated with a physiological glucose concentration by the addition of 40 mm mannitol to the medium. Aortic intima and media from alloxan-diabetic rabbits also exhibit an increased water content and a decreased rate of oxygen uptake. These observations suggest that elevated ambient glucose concentrations result in significant alterations in the metabolism of aortic intima and media.

The Effects of Anoxia on the Morphology and Composite Metabolism of the Intact Aortic Intima-Media Preparation

Paired samples of an intact rabbit aortic intima-media preparation were incubated for short periods under aerobic or anoxic conditions in Krebsbicarbonate buffer containing 6% albumin and 5 mM glucose. During aerobic incubation for as long as 1 h the preparation retained an electron microscopic (EM) appearance similar to that of tissue fixed in situ, and scanning EM confirmed the presence of an uninterrupted endothelial surface. After 2.5 min of anoxia there was widespread endothelial swelling, but the alterations in the EM appearance of these cells were not striking and did not progress during a subsequent 30 min aerobic incubation in fresh medium. After 10 min of anoxia there were marked and widespread alterations in endothelial cell structure, including loss of cell integrity, and numerous discrete interruptions in the endothelium were consistently observed on both transmission and scanning EM. After a subsequent 30 min aerobic incubation in fresh buffer, a major fraction of the luminal surface was denuded of endothelium. The aortic vascular smooth muscle cells did not exhibit evidence of irreversible anoxic injury after 2.5 or 10 min of anoxia or after subsequent aerobic incubation for 30 min. Exposure to anoxia for 10 min induced persistent alterations in the composite metabolism of the preparation during subsequent aerobic incubation in fresh medium; O(2) uptake was reduced, and the fraction of the glucose uptake that was accounted for by lactate production increased approximately 100%. The observations suggest that aortic endothelial cells are dependent upon respiration for the preservation of normal ultrastructure and cell integrity, and probably derive the major fraction of their energy requirements from reactions linked to respiration. Under the conditions employed in these experiments, short periods of anoxia did not induce EM evidence of irreversible anoxic injury in aortic vascular smooth muscle cells; this negative result is not incompatible with other data suggesting that these cells normally derive the major fraction of their energy requirements from respiration. Aortic intima-media does not exhibit a high rate of aerobic glycolysis under aerobic conditions which preserve a normal EM appearance of the preparation, but this pattern of metabolism can be induced by prior anoxic exposure.

The Role of the Polyol Pathway in Methaemoglobin Reduction in Human Red Cells

Summary. The relationship of methaemoglobin reduction to the polyol or sorbitol pathway, which accounts for 2–3% of glucose metabolism at physiological glucose concentrations in human red cells, was investigated. In partially methaemoglobinized cells (3–7% methaemoglobin), the metabolic alterations previously observed at high glucose concentrations did not occur. Partial inhibition of the polyol pathway with 3.3‐tetramethyleneglutaric acid (TMG) resulted in an increase in the pyruvate concentration when compared to non‐treated cells with the same percentage of methaemoglobin. When methaemoglobin production was totally inhibited with carbon monoxide, the metabolic alterations previously observed at a glucose concentration of 50 mm occurred at physiological glucose concentrations. These data suggest that the NADH produced by the polyol pathway may provide reduced pyridine nucleotide required for physiological rates of methaemoglobin reduction.

Gluconeogenesis in toad urinary bladder.

Abstract 1. 1. Toad urinary hemibladders preincubated without added substrate for 18 h contain significant quantities of free glucose and release glucose into the media during subsequent incubations. The addition of lactate, pyruvate, or dihydroxyacetone to the medium increased the rate of glucose release, whereas glutamine and oxaloacetate were without effect. Lactate increased the tissue glycogen content, and 14C from [U-14C]lactate was incorporated into glycogen. Extracellular acidosis increased the rate of glucose release by hemibladders incubated without added substrate or with lactate, but did not increase glucose release by tissue incubated with dihydroxyacetone. 2. 2. Aldosterone increased both the rate of glucose release and the glycogen content of hemibladders incubated with lactate, but equimolar cortisol did not reproduce these effects. Vasopressin acutely increased the rate of glucose release from tissue incubated w without added substrate but not in the presence of lactate. 3. 3. Glucose-6-phosphatase and fructose-1,6-diphosphatase activities were demonstrated in toad urinary bladder. The recovery of 14C from [14C]bicarbonate in glycogen and medium glucose with lactate as substrate suggests that gluconeogenesis in this tissue does not involve the direct reversal of the pyruvate kinase reaction.

Sorbitol synthesis in isolated rat pancreatic islets

Abstract Sorbitol and free fructose are present in isolated rat pancreatic islets in concentrations exceeding that found in plasma or acinar tissues. The sorbitol concentration of the islet increases when the tissue is incubated with increasing glucose concentrations. This suggests a mechanism for the production of the “hydropic” changes observed in the pancreatic islet in hyperglycemic states.

Polyol Pathway Activity in Aorta

Publisher Summary This chapter discusses the polyol pathway activity in aorta. Insulin in vitro has no effect on the glucose space in aortic intima and media, and free intracellular glucose can be demonstrated in tissue from normal or alloxan-diabetic rabbits. Aortic tissue incubated in a medium containing glucose releases free fructose into the medium. The rate of fructose release can be used to provide a minimal estimate of glucose utilization via the polyol pathway. There is a significant turnover of sorbitol in aortic intima and media. Although the steady-state sorbitol concentration in aortic tissue incubated with 5 mM glucose is only 12.9 ± 0.87 nmoles/g, glucose utilization through the polyol pathway accounts for approximately 1.8% of the glucose uptake. Increasing the medium glucose concentration to 50 mM results in increased steady-state sorbitol and fructose concentrations and a sixfold increase in flux through the polyol pathway. The increased sorbitol concentration observed in aortic tissue exposed to elevated glucose concentrations reflects increased sorbitol synthesis.

The Sorbitol Pathway of the Human Erythrocyte

The polyol pathway appears to be widely distributed in human tissues. In this pathway glucose is reduced to its polyol derivative, sorbital, by aldose reductase and the sorbitol then converted to fructose. NADPH seves as a co-factor for glucose reduction while NAD is a co-factor in the second step. The Km for glucose of aldose reductase is sufficiently high so that the intracellular glucose concentration regulates the rate of sorbitol and fructose synthesis. Our studies now demonstrate the presence of a sorbitol pathway in the red cell and its dependence on media of varying glucose concentrations (2–50 mM) and red cell and supernatant sorbitol and fructose measured after incubation. Red cell sorbitol and fructose rose as glucose concentration increased and fructose appeared in the media. Red cell sorbitol rose from 16.0 to 99.6 μmoles/ml as media glucose rose from 2 to 50 mM. Associated with this increase was a rise in the cell lactate to pyruvate ration (69:1 to 193:1), an increase in the percent of glucose metabolized to CO2, and an accumulation of triose phosphates and a fall of the DPG within the cell. These alterations appear to reflect changes in the pyrudine nuclcotide ratios within the cell secondary to the increased metabolism of glucose to fructose. AT high glucose concentration a significant fraction of all glucose metabolized traverses this pathway and is the first evidence that high plasma glucose concentration serves to regulate red cell metabolism. In vivo confirmation of the regulatory role of plasma glucose was provided by the demonstration of increased red cell sorbitol in patients with diabetes and hyperglycemia.