Optimal Duration of Experimental Period in Measurement of Local Cerebral Glucose Utilization with the Deoxyglucose Method

The time course and magnitude of the effects of product loss on the measurement of local cerebral glucose utilization (LCGU) by the 2-[14C]deoxyglucose (DG) method were studied by determination of LCGU in 38 rats with 25-120 min experimental periods after a [14C]DG pulse and in 45 rats with experimental periods of 2.5-120 min during which arterial plasma [14C]DG concentrations (C*P) were maintained constant. LCGU was calculated by the operational equation, which assumes no product loss, with the original set of rate constants and with a new set redetermined in the rats used in the present study; in each case the rate constants were those specific to the structure. Data on local tissue 14C concentrations and C*P were also plotted according to the multiple time/graphic evaluation technique ("Patlak Plot"). The results show that with both pulse and constant arterial inputs of [14C]DG the influence of the rate constants is critical early after onset of tracer administration but diminishes with time and becomes relatively minor by 30 min. After a [14C]DG pulse calculated LCGU remains constant between 25 and 45 min, indicating a negligible effect of product loss during that period; at 60 min it begins to fall and declines progressively with increasing time, indicating that product loss has become significant. When C*P is maintained constant, calculated LCGU does not change significantly over the full 120 min. The "Patlak Plots" reinforced the conclusions drawn from the time courses of calculated LCGU; evidence for loss of product was undetectable for at least 45 min after a pulse of [14C]DG and for at least 60 min after onset of a constant arterial input of [14C]DG.     

Acid Lability of Metabolites of 2-Deoxyglucose in Rat Brain: Implications for Estimates of Kinetic Parameters of Deoxyglucose Phosphorylation and Transport Between Blood and Brain

The steady-state brain/plasma distribution ratios of [14C]deoxyglucose ([14C]DG) for hypoglycemic rats previously determined by measurement of DG concentrations in neutralized acid extracts of freeze-blown brain and plasma exceeded those predicted by simulations of kinetics of the DG model. Overestimation of the true size of the precursor pool of [14C]DG for transport and phosphorylation could arise from sequestration of [14C]DG within brain compartments and/or instability of metabolites of [14C]DG and regeneration of free [14C]DG during the experimental period or extraction procedure. In the present study, the concentrations of [14C]DG and glucose were compared in samples of rat brain and plasma extracted in parallel with perchloric acid or 65% ethanol containing phosphate-buffered saline. The concentrations of both hexoses in acid extracts of brain were higher than those in ethanol, whereas hexose contents of plasma were not dependent on the extraction procedure. The magnitude of overestimation of DG content (about 1.2-to fourfold) varied with glucose level and was highest in extracts isolated from hypoglycemic rats; contamination of the [14C]DG fraction with 14C-labeled nonacidic metabolites also contributed to this overestimation. Glucose concentrations in acid extracts of brain exceeded those of the ethanol extracts by less than 40% for normal and hypoglycemic rats.     

Tissue glucose utilization during epinephrine-induced hyperglycemia

The aim of this study was to investigate glucose utilization by individual tissues during epinephrine infusion. First, the applicability of the 2-deoxyglucose (2-DG) tracer technique during in vivo hyperglycemia was investigated in model systems in vitro. Epitrochlearis muscle and spleen cells were incubated with 1.25-20 mM glucose. The discrimination against 2-[14C]DG in glucose metabolic pathways, expressed by the lumped constant, remained unchanged over this wide range of glucose concentrations. It was concluded that in vivo hyperglycemia does not preclude the application of the 2-DG method. In a series of in vivo experiments, chronically catheterized conscious rats fasted for 24 h and were infused with epinephrine (0.2 microgram.kg-1.min-1), which produced a two-fold increase in plasma glucose concentration. 2-[14C]DG was injected 30 min after starting the epinephrine infusion and glucose utilization rates of individual tissues were calculated based on the concentration of phosphorylated 2-DG in samples excised at 70 min. The epinephrine infusion increased glucose utilization rates by 40-160% in hindlimb muscles, skin, ileum, liver, spleen, lung, epididymal fat, and kidney, although no change was found in the brain. Mass action of the increased plasma glucose is likely to play an important role in the enhanced rate of glucose utilization.     

Tetraethylammonium and p-aminohippurate as clearance markers for renal plasma flow in the rat during saline and glucose infusion.

Renal plasma clearances (C) of 14C-tetraethylammonium (TEA) and p-aminohippurate (PAH) as estimates of arterial renal plasma flow (ARPF) were evaluated in anesthetized rats during control conditions and during intravenous glucose infusion. Venous renal blood flow was measured directly by means of a servo-controlled pump, keeping the renal venous pressure constant. Arteriovenous extraction fractions (E = 1 - P(renal venous)/P(renal arterial)) for PAH averaged 88.3 +/- (SE) 0.8% in control rats and 82.0 +/- 0.9% in glucose-infused rats (p less than 0.001); E(TEA) averaged 92.0 +/- 0.6 and 90.1 +/- 0.6%, respectively (p less than 0.05). Under both experimental conditions, (C/E)PAH did not differ significantly from ARPF, while (C/E)TEA underestimated ARPF; the rate of extraction of TEA exceeded the rate of excretion by 15-20%, probably due to accumulation of TEA in renal tissue. It is concluded that, when corrected for E, C(PAH) is in general a more accurate estimate for ARPF than C(TEA). However, under conditions involving changes in plasma glucose levels C(TEA) may provide a better estimate of the effective renal plasma flow than C(PAH).     

Direct measurement of the lumped constant for 2-deoxy-[1-(14)C]glucose in vivo in human skeletal muscle

The lumped constant (LC) is used to convert the clearance rate of 2-deoxy-D-glucose (2-DG(CR)) to that of glucose (Glc(CR)). There are currently no data to validate the widely used assumption of an LC of 1.0 for human skeletal muscle. We determined the LC for 2-deoxy-[1-(14)C]glucose (2-DG) in 18 normal male subjects (age, 29+/- 2 yr; body mass index, 24.8+/-0.8 kg/m(2)) after an overnight fast and during physiological (1 mU x kg(-1) x min(-1) insulin infusion for 180 min) and supraphysiological (5 mU x kg(-1) x min(-1) insulin infusion for 180 min) hyperinsulinemic conditions. Normoglycemia was maintained with the euglycemic clamp technique. The LC was measured directly with the use of a novel triple tracer-based method. [3-(3)H]glucose, 2-[1-(14)C]DG, and [(12)C]mannitol (Man) were injected as a bolus into the brachial artery. The concentrations of [3-(3)H]glucose and 2-[1-(14)C]DG (dpm/ml plasma) and of Man (micromol/l) were determined in 50 blood samples withdrawn from the ipsilateral deep forearm vein over 15 min after the bolus injection. The LC was calculated by a formula involving blood flow calculated from Man and the Glc(CR) and 2-DG(CR). The LC averaged 1.26+/-0.08 (range 1.06-1.43), 1.15+/-0.05 (0.99-1.39), and 1.18+/-0.05 (0.97-1.37) under fasting conditions and during the 1 and 5 mU x kg(-1). min(-1) insulin infusions (not significant between the different insulin concentrations, mean LC = 1.2, P<0.01 vs. 1.0). We conclude that, in normal subjects, the LC for 2-DG in human skeletal muscle is constant over a wide range of insulin concentrations and averages 1. 2.     

Facilitated Transport of Glucose from Blood into Peripheral Nerve

D-Glucose is the major substrate for energy metabolism in peripheral nerve. The mechanism of transfer of glucose across the blood-nerve barrier is unclarified. In this study an in situ perfusion technique was utilized, in anesthetized rats, to examine monosaccharide transport from blood into peripheral nerve. Unidirectional influxes of D-[14C]glucose, L-[14C]glucose, and [14C]3-O-methyl-D-glucose across capillaries of the tibial nerve were measured at different perfusate concentrations of unlabelled D-glucose. The permeability-surface area product (PA) for D-[14C]glucose and [14C]3-O-methyl-D-glucose decreased, whereas the PA for L-[14C]glucose remained constant, as the perfusate concentration of D-glucose was increased. In the presence of no added unlabelled D-glucose in the perfusate, the PA for L-[14C]glucose equaled one-fifth the PA for D-[14C]glucose. These results demonstrate self-saturation, competitive inhibition, and stereospecificity of glucose transfer, and for the first time show a unidirectional facilitated transport mechanism for D-monosaccharides at capillaries of mammalian peripheral nerve. The data were fit to a model for facilitated transport and passive diffusion. The half-saturation constant and maximal rate of transport for the saturable component of D-glucose influx equaled 23 +/- 11 mumol X ml-1 and 6.6 +/- 3.2 X 10(-3) mumol X s-1 X g-1, respectively. The constant of nonsaturable glucose influx equaled 0.5 +/- 0.1 X 10(-4) s-1. At normal plasma glucose concentrations, the saturable component comprises about 80% of total D-glucose influx into nerve.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucose metabolism in the developing rat. Studies in vivo

1. The specific radioactivity of plasma d-glucose and the incorporation of (14)C into plasma l-lactate, liver glycogen and skeletal-muscle glycogen was measured as a function of time after the intraperitoneal injection of d-[6-(14)C]glucose and d-[6-(3)H]glucose into newborn, 2-, 10- and 30-day-old rats. 2. The log of the specific radioactivity of both plasma d-[6-(14)C]- and d-[6-(3)H]-glucose of the 2-, 10- and 30-day-old rats decreased linearly with time for at least 60min after injection of labelled glucose. The specific radioactivity of both plasma d-[6-(14)C]- and d-[6-(3)H]-glucose of the newborn rat remained constant for at least 75min after injection. 3. The glucose turnover rate of the 30-day-old rat was significantly greater than (approximately twice) that of the 2- and 10-day-old rats. The relative size of both the glucose pool and the glucose space decreased with age. Less than 10% of the glucose utilized in the 2-, 10- and 30-day-old rats was recycled via the Cori cycle. 4. The results are discussed in relationship to the availability of dietary glucose and other factors that may influence glucose metabolism in the developing rat.     

THE [14C]DEOXYGLUCOSE METHOD FOR THE MEASUREMENT OF LOCAL CEREBRAL GLUCOSE UTILIZATION: THEORY,PROCEDURE, AND NORMAL VALUES IN THE CONSCIOUS AND ANESTHETIZED ALBINO RAT1

Abstract— A method has been developed for the simultaneous measurement of the rates of glucose consumption in the various structural and functional components of the brain in vivo. The method can be applied to most laboratory animals in the conscious state. It is based on the use of 2-deoxy-D-[¹⁴C]glucose ([¹⁴C]DG) as a tracer for the exchange of glucose between plasma and brain and its phosphorylation by hexokinase in the tissues. [¹⁴C]DG is used because the label in its product, [¹⁴C]deoxyglucose-6-phosphate, is essentially trapped in the tissue over the time course of the measurement. A model has been designed based on the assumptions of a steady state for glucose consumption, a first order equilibration of the free [¹⁴C]DG pool in the tissue with the plasma level, and relative rates of phosphorylation of [¹⁴C]DG and glucose determined by their relative concentrations in the precursor pools and their respective kinetic constants for the hexokinase reaction. An operational equation based on this model has been derived in terms of determinable variables. A pulse of [¹⁴C]DG is administered intravenously and the arterial plasma [¹⁴C]DG and glucose concentrations monitored for a preset time between 30 and 45min. At the prescribed time, the head is removed and frozen in liquid N₂-chilled Freon XII, and the brain sectioned for autoradiography. Local tissue concentrations of [¹⁴C]DG are determined by quantitative autoradiography. Local cerebral glucose consumption is calculated by the equation on the basis of these measured values. The method has been applied to normal albino rats in the conscious state and under thiopental anesthesia. The results demonstrate that the local rates of glucose consumption in the brain fall into two distinct distributions, one for gray matter and the other for white matter. In the conscious rat the values in the gray matter vary widely from structure to structure (54-197 μmol/100 g/min) with the highest values in structures related to auditory function, e.g. medial geniculate body, superior olive, inferior colliculus, and auditory cortex. The values in white matter are more uniform (i.e. 33–40 μmo1/100 g/min) at levels approximately one-fourth to one-half those of gray matter. Heterogeneous rates of glucose consumption are frequently seen within specific structures, often revealing a pattern of cytoarchitecture. Thiopental anesthesia markedly depresses the rates of glucose utilization throughout the brain, particularly in gray matter, and metabolic rate throughout gray matter becomes more uniform at a lower level.     

Negligible glucose-6-phosphatase activity in cultured astroglia

2-Deoxy[14C]glucose-6-phosphate (2-[14C]DG-6-P) dephosphorylation and glucose-6-phosphatase (G-6-Pase) activity were examined in cultured rat astrocytes under conditions similar to those generally used in assays of glucose utilization. Astrocytes were loaded with 2-[14C]DG-6-P by preincubation for 15 min in medium containing 2 mM glucose and 50 microM 2-deoxy[14C]glucose (2-[14C]DG). The medium was then replaced with identical medium including 2 mM glucose but lacking 2-[14C]DG, and incubation was resumed for 5 min to diminish residual free 2-[14C]DG levels in the cells by either efflux or phosphorylation. The medium was again replaced with fresh 2-[14C]DG-free medium, and the incubation was continued for 5, 15, or 30 min. Intracellular and extracellular 14C contents were measured at each time point, and the distribution of 14C between 2-[14C]DG and 2-[14C]DG-6-P was characterized by paper chromatography. The results showed little if any hydrolysis of 2-[14C]DG-6-P or export of free 2-[14C]DG from cells to medium; there were slightly increasing losses of 2-[14C]DG and 2-[14C]DG-6-P into the medium with increasing incubation time, but they were in the same proportions found in the cells, suggesting they were derived from nonadherent or broken cells. Experiments carried out with medium lacking glucose during the assay for 2-deoxyglucose-6-phosphatase activity yielded similar results. Evidence for G-6-Pase activity was also sought by following the selective detritiation of glucose from the 2-C position when astrocytes were incubated with [2-3H]glucose and [U-14C]glucose in the medium. No change in the 3H/14C ratio was found in incubations for as long as 15 min. These results indicate negligible G-6-Pase activity in cultured astrocytes.     

Blood glycerol is an important precursor for intramuscular triacylglycerol synthesis.

The utilization of blood glycerol and glucose as precursors for intramuscular triglyceride synthesis was examined in rats using an intravenous infusion of [2-(14)C]glycerol and [6-(3)H]glucose or [6-(14)C]glucose. In 24-h fasted rats, more glycerol than glucose was incorporated into intramuscular triglyceride glycerol in soleus (69 +/- 23 versus 4 +/- 1 nmol/micromol triglyceride/h, respectively, p = 0.02 glycerol versus glucose) and in gastrocnemius (25 +/- 5 versus 9 +/- 2 nmol/micromol triglyceride/h, respectively, p = 0.02). Blood glucose was utilized more than blood glycerol for triglyceride glycerol synthesis in quadriceps. In fed rats, the blood glycerol incorporation rates (4 +/- 2, 8 +/- 3, and 9 +/- 3 nmol/micromol triglyceride/h) were similar (p > 0.3) to those of glucose (5 +/- 2, 8 +/- 2, and 5 +/- 2 nmol/micromol triglyceride/h for quadriceps, gastrocnemius, and soleus muscle, respectively). Glucose incorporation into intramuscular triglycerides was less with [6-(3)H]glucose than with [6-(14)C]glucose, suggesting an indirect pathway for glucose carbon entry into muscle triglyceride. The isotopic equilibrium between plasma and intramuscular free glycerol ([U-(13)C]glycerol) was complete in quadriceps and gastrocnemius, but not soleus, within 2 h after beginning the tracer infusion. We conclude that blood glycerol is a direct and important precursor for muscle triglyceride synthesis in rats, confirming the presence of functionally important amounts of glycerol kinase in skeletal muscle.     

Contribution of propionate to glucose synthesis in sheep

1. The production rate of propionate in the rumen and the entry rate of glucose into the body pool of glucose in sheep were measured by isotope-dilution methods. Propionate production rates were measured by using a continuous infusion of specifically labelled [(14)C]propionate. Glucose entry rates were estimated by using either a primed infusion or a continuous infusion of [U-(14)C]glucose. 2. The specific radioactivity of plasma glucose was constant between 4 and 9hr. after the commencement of intravenous infusion of [U-(14)C]glucose and between 1 and 3hr. when a primed infusion was used. 3. Infusion of [(14)C]propionate intraruminally resulted in a fairly constant specific radioactivity of rumen propionate between about 4 and 9hr. and of plasma glucose between 6 and 9hr. after the commencement of the infusion. Comparison of the mean specific radioactivities of glucose and propionate during these periods allowed estimates to be made of the contribution of propionate to glucose synthesis. 4. Comparisons of the specific radioactivities of plasma glucose and rumen propionate during intraruminal infusions of one of [1-(14)C]-, [2-(14)C]-, [3-(14)C]- and [U-(14)C]-propionate indicated considerable exchange of C-1 of propionate on conversion into glucose. The incorporation of C-2 and C-3 of propionate into glucose and lactate indicated that 54% of both the glucose and lactate synthesized arose from propionate carbon. 5. No differences were found for glucose entry rates measured either by a primed infusion or by a continuous infusion. The mean entry rate (+/-s.e.m.) of glucose estimated by using a continuous infusion into sheep was 0.33+/-0.03 (4) m-mole/min. and by using a primed infusion was 0.32+/-0.01 (4) m-mole/min. The mean propionate production rate was 1.24+/-0.03 (8) m-moles/min. The conversion of propionate into glucose was 0.36 m-mole/min., indicating that 32% of the propionate produced in the rumen is used for glucose synthesis. 6. It was indicated that a considerable amount of the propionate converted into glucose was first converted into lactate.     

Competition among oxidizable substrates in brains of young and adult rats. Dissociated cells.

The rates of conversion of D-(-)-3-hydroxy[3-14C]butyrate, [3-14C]acetoacetate, [6-14C]glucose and [U-14C]glutamine into 14CO2 were measured in the presence and absence of alternative oxidizable substrates in intact dissociated cells from the brains of young and adult rats. When unlabelled glutamine was added to [6-14C]glucose or unlabelled glucose was added to [U-14C]glutamine, the rate of 14CO2 production was decreased in both young and adult rats. The rate of oxidation of 3-hydroxy[3-14C]butyrate was also decreased by the addition of unlabelled glutamine in both age groups, but in the reverse situation, i.e. unlabelled 3-hydroxybutyrate added to [U-14C]glutamine, only the brain cells from young rats were affected. No significant effects were seen when glutamine and acetoacetate were combined. The addition of either of the two ketone bodies to [6-14C]glucose markedly lowered the rate of 14CO2 production in young rats, but in the adult only 3-hydroxybutyrate was effective and the magnitude of decrease in the rate of [6-14C]glucose oxidation was much lower than in young animals. Unlabelled glucose decreased the rate of [3-14C]acetoacetate oxidation to a minor extent in brain cells from both age groups; when added to 3-hydroxy[3-14C]butyrate, glucose had no effect in young rats and greatly enhanced 14CO2 production in adult brain cells. Many of these patterns of substrate interaction in dissociated brain cells differ from those in whole homogenates; they may be a function of the plasma membranes and the role of a carrier-mediated transport system or a reflection of a difference in the population of cell types or subcellular organelles in these two preparations.     

Regulation of myocardial [(13)C]glucose metabolism in conscious rats

Administration of supplemental glucose and/or insulin is postulated to improve the outcome from myocardial ischemia by increasing the heart's relative utilization of glucose as an energy substrate. To examine the degree to which circulating glucose and insulin levels actually influence myocardial substrate preference in vivo, we infused conscious, chronically catheterized rats with D-[1-(13)C]glucose and compared steady-state (13)C enrichment of plasma glucose with that of myocardial glycolytic ([3-(13)C]alanine) and oxidative ([4-(13)C]glutamate) intermediary metabolites. In fasting rats, [3-(13)C]alanine-to-[1-(13)C]glucose and [4-(13)C]glutamate-to-[3-(13)C]alanine ratios averaged 0.16 +/- 0.12 and 0.14 +/- 0.03, respectively, indicating that circulating glucose contributed 32% of myocardial glycolytic flux, whereas subsequent flux through pyruvate dehydrogenase contributed 14% of total tricarboxylic acid (TCA) cycle activity. Raising plasma glucose to 11 mmol/l, or insulin to 500 pmol/l, increased these contributions equivalently. At supraphysiological (>6,500 pmol/l) insulin levels, the plasma glucose contribution to glycolysis increased further, and addition of hyperglycemia made it the sole glycolytic substrate, yet [4-(13)C]glutamate-to-[3-(13)C]alanine ratios remained /=40% of myocardial TCA cycle flux.     

Fiber type-specific determinants of Vmax for insulin-stimulated muscle glucose uptake in vivo

The aim of this study was to determine barriers limiting muscle glucose uptake (MGU) during increased glucose flux created by raising blood glucose in the presence of fixed insulin. The determinants of the maximal velocity (V(max)) of MGU in muscles of different fiber types were defined. Conscious rats were studied during a 4 mU x kg(-1) x min(-1) insulin clamp with plasma glucose at 2.5, 5.5, and 8.5 mM. [U-(14)C]mannitol and 3-O-methyl-[(3)H]glucose ([(3)H]MG) were infused to steady-state levels (t = -180 to 0 min). These isotope infusions were continued from 0 to 40 min with the addition of a 2-deoxy-[(3)H]glucose ([(3)H]DG) infusion. Muscles were excised at t = 40 min. Glucose metabolic index (R(g)) was calculated from muscle-phosphorylated [(3)H]DG. [U-(14)C]mannitol was used to determine extracellular (EC) H(2)O. Glucose at the outer ([G](om)) and inner ([G](im)) sarcolemmal surfaces was determined by the ratio of [(3)H]MG in intracellular to EC H(2)O and muscle glucose. R(g) was comparable at the two higher glucose concentrations, suggesting that rates of uptake near V(max) were reached. In summary, by defining the relationship of arterial glucose to [G](om) and [G](im) in the presence of fixed hyperinsulinemia, it is concluded that 1) V(max) for MGU is limited by extracellular and intracellular barriers in type I fibers, as the sarcolemma is freely permeable to glucose; 2) V(max) is limited in muscles with predominantly type IIb fibers by extracellular resistance and transport resistance; and 3) limits to R(g) are determined by resistance at multiple steps and are better defined by distributed control rather than by a single rate-limiting step.     

Comparative glucose utilization rates in separated and mated schistosomes

The rate of phosphorylation of 2-deoxyglucose (2DG) was determined by sequential pulsing of schistosomes (Schistosoma mansoni, S. japonicum, and S. haematobium) with 3H- and 14C-labeled 2-deoxy-D-glucose. Subsequent column chromatographic separation of the neutral [3H]2DG and [14C]2DG from the 3H- and 14C-labeled 2-deoxy-D-glucose 6-phosphate permitted estimation of the quantity of [3H]2DG phosphorylated in 2 min, and the proportion of [14C]2DG phosphorylated in 1 min; thus a phosphorylation rate was determined from a single tissue sample. The relative phosphorylation rate of 2-[3H]2DG to D-1-[14C]glucose (i.e., the phosphorylation coefficient) was also measured in male and female schistosomes. It was demonstrated that even though 2DG is taken up more rapidly than glucose, it is phosphorylated at a much slower rate in both S. mansoni and S. japonicum. In both of these species, mated males phosphorylate 2DG and glucose at a greater rate than do unmated males. Similarly, mated females phosphorylate and consume more glucose than do separated females. In contrast, the phosphorylation coefficient is greater in separated than in mated schistosomes. Intraspecific comparisons suggest that, at reduced substrate concentrations, glucose utilization rates are higher in S. japonicum, intermediate in S. mansoni, and lower in S. haematobium.     

The distribution of glucose and [14C]glucose between erythrocytes and plasma in the rat

1. Of the glucose in rat blood 79.8+/-3.3% (s.d.) was in the plasma. The variance was mostly due to differences between rats. 2. The concentration of glucose in erythrocyte water was 51+/-8% (s.d.) of that in plasma water. 3. The ratio (specific radioactivity in plasma)/(specific radioactivity in whole blood), i.e. the P/B ratio, was estimated for glucose at intervals after intravenous injection of [U-(14)C]glucose and [U-(14)C]fructose. The ratio differed from unity by more than the standard error of a single determination of the specific radioactivity of blood or plasma glucose except from 10 to 17min. after injection of [(14)C]glucose and from 22 to 30min. after injection of [(14)C]fructose. At all other times specific radioactivities in blood had to be corrected to give specific radioactivities in plasma. How to do so is described. 4. The P/B ratios were accounted for by a turnover of glucose in erythrocytes of 0.14mumole/min./ml. of erythrocytes. 5. Metabolism of glucose in rat erythrocytes is unlikely to be a major source of lactate.     

Effect of dihydroxyacetone and pyruvate on plasma glucose concentration and turnover in noninsulin-dependent diabetes mellitus

Consumption of dihydroxyacetone and pyruvate (DHP) increases muscle extraction of glucose in normal men. To test the hypothesis that these three-carbon compounds would improve glycemic control in diabetes, we evaluated the effect of DHP on plasma glucose concentration, turnover, recycling, and tolerance in 7 women with noninsulin-dependent diabetes. The subjects consumed a 1,500-calorie diet (55% carbohydrate, 30% fat, 15% protein), randomly containing 13% of the calories as DHP (1/1) or Polycose (placebo; PL), as a drink three times daily for 7 days. On the 8th day, primed continuous infusions of [6-3H]-glucose and [U-14C]-glucose were begun at 05.00 h, and at 09.00 h a 3-hour glucose tolerance test (75 g glucola) was performed. Two weeks later the subjects repeated the study with the other diet. The fasting plasma glucose level decreased by 14% with DHP (DHP = 8.0 +/- 0.9 mmol/l; PL = 9.3 +/- 1.0 mmol/l, p less than 0.05) which accounted for lower postoral glucose glycemia (DHP = 13.1 +/- 0.8 mmol/l, PL = 14.7 +/- 0.8 mmol/l, p less than 0.05). [6-3H]-glucose turnover (DHP = 1.50 +/- 0.19 mg.kg-1.min-1, PL = 1.77 +/- 0.21 mg.kg-1.min-1, p less than 0.05) and glucose recycling, the difference in [6-3H]-glucose and [U-14C]-glucose turnover rates, decreased with DHP (DHP = 0.25 +/- 0.07 mg.kg-1.min-1, PL = 0.54 +/- 0.10 mg.kg-1.min-1, p less than 0.05). Fasting and postoral glucose, plasma insulin, glucagon, and C peptide levels were unaffected by DHP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of Glucose Utilization Rates in Cultured Astrocytes and Neurons with [<Superscript>14</Superscript>C]deoxyglucose: Progress,Pitfalls, and Discovery of Intracellular Glucose Compartmentation

2-Deoxy-d-[¹⁴C]glucose ([¹⁴C]DG) is commonly used to determine local glucose utilization rates (CMR_glc) in living brain and to estimate CMR_glc in cultured brain cells as rates of [¹⁴C]DG phosphorylation. Phosphorylation rates of [¹⁴C]DG and its metabolizable fluorescent analog, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), however, do not take into account differences in the kinetics of transport and metabolism of [¹⁴C]DG or 2-NBDG and glucose in neuronal and astrocytic cells in cultures or in single cells in brain tissue, and conclusions drawn from these data may, therefore, not be correct. As a first step toward the goal of quantitative determination of CMR_glc in astrocytes and neurons in cultures, the steady-state intracellular-to-extracellular concentration ratios (distribution spaces) for glucose and [¹⁴C]DG were determined in cultured striatal neurons and astrocytes as functions of extracellular glucose concentration. Unexpectedly, the glucose distribution spaces rose during extreme hypoglycemia, exceeding 1.0 in astrocytes, whereas the [¹⁴C]DG distribution space fell at the lowest glucose levels. Calculated CMR_glc was greatly overestimated in hypoglycemic and normoglycemic cells because the intracellular glucose concentrations were too high. Determination of the distribution space for [¹⁴C]glucose revealed compartmentation of intracellular glucose in astrocytes, and probably, also in neurons. A smaller metabolic pool is readily accessible to hexokinase and communicates with extracellular glucose, whereas the larger pool is sequestered from hexokinase activity. A new experimental approach using double-labeled assays with DG and glucose is suggested to avoid the limitations imposed by glucose compartmentation on metabolic assays.     

Postnatal changes in dolichol-pathway enzyme activities in rat liver.

The activity of hepatic protein N-glycosylation was compared in rats of different ages by incubating UDP-[14C]glucose with liver microsomes. Dolichyl-phosphate [14C]glucose, [14C]glucosyl-oligosaccharide-lipid and [14C]glycoproteins formed were increased after birth to maximal levels at 2 weeks; thereafter dolichylphosphate [14C]glucose remained constant, while [14C]glucosyl-oligosaccharide-lipid and [14C]glycoproteins were decreased to constant levels at 4 weeks. The postnatal change in the formation of [14C]glycoproteins was similar to the change in the hexosamine content of N-glycans in liver microsomes and plasma, suggesting that the N-glycosylation of proteins in rat liver increases after birth to a maximum at 2 weeks, and thereafter decreases to a constant level at 4 weeks. The possibility of a regulatory role for dolichyl phosphate in glycoprotein synthesis in rat liver during postnatal development was eliminated by demonstrating the inefficiency of exogenous dolichyl phosphate on the postnatal changes in [14C]glycoprotein formation. The transfer of [14C]glucose from UDP-[14C]glucose to denatured alpha-lactalbumin in liver microsomes increased to a maximum at 2 weeks and then decreased to a constant level, as with transfer to endogenous proteins (i.e. the formation of [14C]glycoproteins). On the other hand, the transfer of oligosaccharide from exogenous [14C]glucosyl-oligosaccharide-lipid to denatured alpha-lactalbumin reached a maximum at 2 weeks and then remained constant. These results strongly suggest that oligosaccharide-lipid available for N-glycosylation is limiting in rat liver after 2 weeks post partum. The activities of dolichyl-phosphate glucose, dolichyl-phosphate mannose and dolichyl-pyrophosphate N-acetylglucosamine synthases increased until 2 weeks post partum. Thereafter, the activity of dolichyl-pyrophosphate N-acetylglucosamine synthase decreased to a constant level at 4 weeks, while the activities of dolichyl-phosphate glucose and dolichyl-phosphate mannose synthases remained constant. These results suggest that N-glycosylation of proteins in rat liver increases until 2 weeks post partum, and that this depends on the activities of dolichol-pathway enzymes as a whole rather than on the activity of specific enzymes. N-Glycosylation then decreases to a constant level at 4 weeks due to decreases in the activities of enzymes responsible for oligosaccharide assembly on lipids, including dolichyl-pyrophosphate N-acetylglucosamine synthase.     

Insulin effect on in vivo gluconeogenesis from [3-14C]pyruvate in the starved rat

During a 24 hr fast rats received 4 subcutaneous injections of insulin, and 15 min after the last injection they were given an intravenous pulse of [3-14C]pyruvate. The amount of [14C]glucose in blood 2 min after the tracer did not differ between insulin treated and control animals, whereas at 5 and 10 min values were significantly lower in the former group. At 10 min after the tracer, liver [14C]glycogen specific activity and [14C]fatty acid amount were higher in the insulin treated animals than in controls while plasma concentration of gluconeogenic amino acids was lower in the first group. Similar changes but less pronounced and more retarded were found in 24 hr fasted rats given only one insulin dose 15 min before the [3-14C]pyruvate pulse. Results indicate that gluconeogenesis from pyruvate is not directly modified by insulin treatment. Effects found at 5 and/or 10 min after the tracer and reported effects after prolonged insulin treatments may be caused by one or all of the following possibilities: enhanced utilization of the new-formed glucose, reduced availability of gluconeogenic substrates, and counteracting action on gluconeogenic hormones.