Albert I. Winegrad

Effects of insulin and dietary myoinositol on impaired peripheral motor nerve conduction velocity in acute streptozotocin diabetes.

The factors influencing the development of impaired sciatic motor nerve conduction velocity (MNCV) in acute experimental diabetes were examined. Decreased MNCV developed by the 14th day after streptozotocin administration but only in rats which became hyperglycemic. Insulin treatment, begun on day 3, failed to prevent imparied MNCV in diabetic rats in which improved or normal weight gain and a decreased degree of hyperglycemia was induced. However, insulin treatment prevented the development of impaired MNCV in a group of diabetic rats in which the tail vein plasma glucose concentration was never found to exceed 160 mg/dl during days 6 through 14, andin which the mean plus or minus SEM of the average plasma glucose concentration for each animal during the same period was 75 plus or minus 18 mg/dl. In normal rats fed diets containing 0.011% or 0.069% free myoinositol (a presumably normal range), sciatic nerve free myoinositol concentrations were 90- and 60-fold higher than those in plasma. On these diets the development of impaired MNCV in the diabetics was associated with a decrease in nerve free myoinositol as compared with nerves from normals fed the same diet, despite similar plasma levels in the normals and diabetics. Plasma and nerve free myoinositol increased with increasing dietary myoinositol content in both normals and diabetics, and nerve myoinositol content could be acutely increased by an i.p. myoinositol load. By supplementing the diets with 1.0% myoinositol, the difference in nerve myoinositol in normal and diabetic rats on day 14 was abolished; on this diet the development of impaired MNCV in the diabetics was moderated or totally prevented, despite persistent hyperglycemia and elevated nerve sorbitol and fructose concentrations. Insulin treatment that prevented impaired MNCV prevented a decrease in nerve myoinositol in diabetics. These studies suggest that insulin deficiency, and possibly hyperglycemia, are primary factors in the development of imparied MNCV in acute experimental diabetes. However, the development of impaired MNCV appears to be related in some manner to a derangement in the regulation of nerve free myoinositol content, which appears to be subject to modification by increases in plasma myoinositol concentration over a critical range.

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.

Effects of Acute Experimental Diabetes on Composite Energy Metabolism in Peripheral Nerve Axons and Schwann Cells

Rabbit tibial nerve fascicle and endoneurial preparations were used to study the effects of alloxan diabetes on composite energy metabolism in peripheral nerve axons and Schwann cells. Glucose was preferentially used for energy provision in fascicle and endoneurial preparations from diabetic rabbits (despite very high endogenous fructose concentrations) and respiration provided the preponderant fraction of their energy requirements. Diabetic fascicles incubated with 20 mM glucose maintained a steady state of energy metabolism for 2 h, as assessed by O2 uptake , ATP levels, and P-creatine/creatine ratio. Their steadystate rate of energy utilization was estimated from the theoretical yield of ATP from the rates of glucose utilization required to sustain the steady state O2 uptake and lactate production. This was equivalent to the use of 117 mmol ATP/kg/h, a value 26% lower than in normal fascicles, but the ATP levels and P-creatine/creatine ratios in diabetic fascicles were unaltered. Diabetic endoneurium maintained a near steady state of energy metabolism when incubated with 5 mM glucose; its rate of energy utilization was 31% lower than in normal endoneurium, although their ATP levels and P-creatine/creatine ratios were similar. Experimental diabetes results in a striking decrease in the composite steady-state rate of energy utilization in axons and Schwann cells, suggesting that the rates of specific energy requiring processes operative in normal nerve are decreased. Diabetic fascicles and endoneurium had significantly decreased myo-inositol and creatine + P-creatine concentrations, suggesting that the maintenance of the normal intracellular/extracellular gradients of myo-inositol and creatine is impaired. The rate of energy utilization in normal endoneurium was not altered by incubation with 20 mM glucose for 2 h. Insulin (100 μU/ml) did not increase the rate of energy utilization in diabetic endoneurium incubated with glucose.

In Vitro Studies of the Substrates for Energy Production and the Effects of Insulin on Glucose Utilization in the Neural Components of Peripheral Nerve

An “endoneurial” preparation from a rabbit tibial nerve fascicle was used to study the ability of peripheral nerve axons and Schwann cells to derive their composite energy requirements from glucose, D-β-hydroxybutyrate, or albumin-bound palmitate, and the effects of insulin in vitro on their composite glucose utilization. Samples incubated with 5 mM glucose for 2 h maintained a stable O2 uptake and P-creatine and ATP concentrations, and they exhibited a slight increase in P-creatine/creatine ratio (the electron microscopic appearance of the preparation was previously shown to be unaltered under these conditions). The rate of glucose oxidation required to account for the O2 uptake accounted for 61% of the glucose uptake. In samples incubated without substrate for 2 h, a marked fall in tissue glucose was associated with a 50% decrease in O2 uptake and with decreases in P-creatine, ATP, and in the P-creatine/creatine ratio. In medium lacking glucose but containing 5 mM DL-β-hydroxybutyrate, a stable rate of D-β-hydroxybutyrate uptake was observed, and acetoacetate production accounted for only a small fraction; significant decreases in O2 uptake or ATP were prevented, and, although P-creatine and the P-creatine/creatine ratio fell, they remained significantly higher than after incubation without substrate. An efficient blood-nerve barrier to albumin is known to exist. Medium containing albumin-bound palmitate with molar ratios of palmitate/albumin of 1 or 2 (highest FFA concentration, 1.32 meq/L) failed to prevent decreases in P-creatine, ATP, and in the P-creatine/creatine ratio during incubations without glucose; the associated O2 uptakes suggested that the tissue is susceptible to respiratory uncoupling and depression on exposure to albumin-bound palmitate as compared with non-neural tissue. Insulin (100 or 1000 μU/ml) had no detectable effects on glucose utilization in the endoneurial preparation during 2-h incubations with 5 mM glucose or (U-14C) glucose. In contrast, in epineurial tissue from rabbit sciatic nerve, insulin (100 μU/ml) increased (U-14C) glucose incorporation into CO2 and total lipid. The neural components of peripheral nerve are probably dependent on glucose as their major substrate for energy production and respiration under most physiologic conditions in which elevated plasma ketone body concentrations are absent; their composite glucose utilization is not subject to acute, direct regulation by insulin in concentrations that might reasonably be derived from plasma insulin of pancreatic origin.

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.

Basal phosphatidylinositol turnover controls aortic Na+/K+ ATPase activity.

To determine whether basal phosphoinositide turnover plays a role in metabolic regulation in resting rabbit aortic intima-media incubated under steady state conditions, we used deprivation of extracellular myo-inositol as a potential means of inhibiting basal phosphatidylinositol (PI) synthesis at restricted sites and of depleting small phosphoinositide pools with a rapid basal turnover. Medium myo-inositol in a normal plasma level was required to prevent inhibition of a specific component of basal de novo PI synthesis that is necessary to demonstrate a discrete rapidly turning-over [1,3-14C]glycerol-labeled PI pool. Medium myo-inositol was also required to label the discrete PI pool with [1-14C]arachidonic acid (AA). The rapid basal turnover of this PI pool, when labeled with glycerol or AA, was not attributable to its utilization for polyphosphoinositide formation, and it seems to reflect basal PI hydrolysis. Depleting endogenous free AA with medium defatted albumin selectively inhibits the component of basal de novo PI synthesis that replenishes the rapidly turning-over PI pool. A component of normal resting energy utilization in aortic intima-media also specifically requires medium myo-inositol in a normal plasma level and a free AA pool; its magnitude is unaltered by indomethacin, nordihydroguaiaretic acid, or Ca2+-free medium. This energy utilization results primarily from Na+/K+ ATPase activity (ouabain-inhibitable O2 consumption), and in Ca2+-free medium deprivation of medium myo-inositol or of free AA inhibits resting Na+/K+ ATPase activity to a similar degree (60%, 52%). In aortic intima-media basal PI turnover controls a major fraction of resting Na+/K+ ATPase activity.

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.

Mechanism of glucose-induced (Na+, K+)-ATPase inhibition in aortic wall of rabbits

SummaryHyperglycaemia decreases (Na+, K+)-ATPase activity in specific tissues by a mechanism whose effects are prevented by aldose reductase inhibitors and by raising plasma myo-inositol. This mechanism was activated and studied in vitro in normal rabbit aortic intima-media. Raising medium glucose to 10 mmol/l for 60 min inhibited a major component of (Na+, K+)-ATPase-mediated 86Rb + /K+ uptake normally operative in resting aortic intima-media in medium containing normal plasma levels of glucose (5 mmol/l) and myo-inositol (70 μmol/l); 20 or 30 mmol/l glucose had no greater effect. This effect occurred under conditions in which the aortic intima-medias normal myo-inositol content is not detectably decreased. The inhibition was prevented by sorbinil (10 μmol/l) and by raising medium myo-inositol from 70 to 500 μmol/l, which had no effect on (Na+, K+)-ATPase activity when the medium glucose remained at 5 mmol/l. Raising medium glucose selectively inhibited a component of (Na+, K+)-ATPase activity that requires medium myo-inositol, because it is maintained by a regulatory system through rapid basal phosphatidylinositol turnover in a discrete pool, which is replenished by a fraction of basal de novo phosphatidylinositol synthesis that is selectively dependent on myo-inositol uptake. Medium myo-inositol at a normal plasma level became inadequate to maintain this fraction of basal de novo phosphatidylinositol synthesis ([1,3-14C]glycerol incorporation) when the medium glucose was raised. When sorbinil was added raising medium glucose did not alter the ability of 70 umol/l medium myoinositol to maintain the (Na+, K+)-ATPase activity that requires medium myo-inositol. The inhibition caused by raising medium glucose was reproduced by a competitive inhibitor of active myo-inositol transport, scyllo-inositol (500 μmol/l). The effect of medium glucose in an elevated plasma level on (Na+, K+)-ATPase activity in aortic intima-media appears to result from increased polyol pathway activity that makes myoinositol uptake at a normal plasma level inadequate to maintain the normal operation of a regulatory system.

Hexokinase Isoenzymes in Human Erythrocytes: Association of Type II with Fetal Hemoglobin

Hexokinase activity in human erythrocytes is associated with three electrophoretically distinct bands. Normal adult erythrocytes contain hexokinases Types I and III. Type II hexokinase is present in the erythrocytes of newborn infants and absent from those of normal adults; it is, however, present in erythrocytes of adults with hereditary persistence of fetal hemoglobin. Type II hexokinase and fetal hemtoglobin appear to be associated.