Interstitial glucose concentration and glycemia: implications for continuous subcutaneous glucose monitoring

The changes in plasma glucose concentration and in interstitial glucose concentration, determined with a miniaturized subcutaneous glucose sensor, were investigated in anesthetized nondiabetic rats. Interstitial glucose was estimated through two different calibration procedures. First, after a glucose load, the magnitude of the increase in interstitial glucose, estimated through a one-point calibration procedure, was 70% of that in plasma glucose. We propose that this is due to the effect of endogenous insulin on peripheral glucose uptake. Second, during the spontaneous secondary decrease in plasma glucose after the glucose load, interstitial glucose decreased faster than plasma glucose, which may also be due to the effect of insulin on peripheral glucose uptake. Third, during insulin-induced hypoglycemia, the decrease in interstitial glucose was less marked than that of plasma glucose, suggesting that hypoglycemia suppressed transfer of glucose into the interstitial tissue; subsequently, interstitial glucose remained lower than plasma glucose during its return to basal value, suggesting that the stimulatory effect of insulin on peripheral glucose uptake was protracted. If these observations obtained in rats are relevant to human physiology, such discrepancies between plasma and interstitial glucose concentration may have major implications for the use of a subcutaneous glucose sensor in continuous blood glucose monitoring in diabetic patients.     

Intracellular Glucose Concentration in Derepressed Yeast Cells Consuming Glucose Is High Enough To Reduce the Glucose Transport Rate by 50%

In Saccharomyces cerevisiae cells exhibiting high-affinity glucose transport, the glucose consumption rate at extracellular concentrations above 10 mM was only half of the zero trans-influx rate. To determine if this regulation of glucose transport might be a consequence of intracellular free glucose we developed a new method to measure intracellular glucose concentrations in cells metabolizing glucose, which compares glucose stereoisomers to correct for adhering glucose. The intracellular glucose concentration was 1.5 mM, much higher than in most earlier reports. We show that for the simplest model of a glucose carrier, this concentration is sufficient to reduce the glucose influx by 50%. We conclude that intracellular glucose is the most likely candidate for the observed regulation of glucose import and hence glycolysis. We discuss the possibility that intracellular glucose functions as a primary signal molecule in these cells.     

Four grams of glucose

Four grams of glucose circulates in the blood of a person weighing 70 kg. This glucose is critical for normal function in many cell types. In accordance with the importance of these 4 g of glucose, a sophisticated control system is in place to maintain blood glucose constant. Our focus has been on the mechanisms by which the flux of glucose from liver to blood and from blood to skeletal muscle is regulated. The body has a remarkable capacity to satisfy the nutritional need for glucose, while still maintaining blood glucose homeostasis. The essential role of glucagon and insulin and the importance of distributed control of glucose fluxes are highlighted in this review. With regard to the latter, studies are presented that show how regulation of muscle glucose uptake is regulated by glucose delivery to muscle, glucose transport into muscle, and glucose phosphorylation within muscle.     

Kinetic studies on glucose and xylose transport in Saccharomyces cerevisiae

Zero trans-influx assays of glucose and xylose were performed using Saccharomyces cerevisiae to investigate transport characteristics under high and low glucose conditions. Under high glucose conditions, most glucose was transported by the low-affinity transporter. The high-affinity transporter was expressed under low glucose conditions, transporting over 50% glucose. Inhibition kinetics revealed that xylose was transported by both high- and low-affinity glucose transporters. Affinities of both glucose transporters for xylose were very low under high glucose condition but increased to a similar level to glucose under low glucose condition. The maximum rate of xylose transport increased by 85%, while an overall maximum glucose transport rate decreased by 42% under low glucose condition, indicating the presence of other transport system for sugars except for glucose. It was suggested that expression of the high-affinity transporter and increased affinity of glucose transporters for xylose under low glucose condition would provide a fermentation strategy for enhancing the productivity of xylitol by recombinant S. cerevisiae harboring the xylose reductase gene.     

Continuous glucose monitoring and control in a rotating wall perfused bioreactor

A glucose control system consisting of a single in-line glucose sensor, concentrated glucose solution, and computer hardware and software were developed. The system was applied to continuously control glucose concentrations of a perfusion medium in a rotating wall perfused vessel (RWPV) bioreactor culturing BHK-21 cells. The custom-made glucose sensor was based on a hydrogen peroxide electrode. The sensor continuously and accurately measured the glucose concentration of GTSF-2 medium in the RWPV bioreactor during cell culture. Three sets of two-point calibrations were applied to the glucose sensor during the 55-day cell culture. The system first controlled the glucose concentration in perfusing medium between 4.2 and 5.6 mM for 36 days and then at different glucose levels for 19 days. A stock solution with a high glucose concentration (266 mM) was used as the glucose injection solution. The standard error of prediction (SEP) for glucose measurement by the sensor, compared to measurement by the Beckman glucose analyzer, was +/-0.4 mM for 55 days.     

The role of cholecystokinin (CCK8) on glucose production and elimination, and on plasma insulin and glucose in rats

The effects of the gastrointestinal hormone and neurotransmitter cholecystokinin (CCK8) are complex, since it exhibits both an insulinotropic and a glucagonotropic effect. We investigated CCK8 in vivo with respect to glucose fluxes (production and elimination) at both low (6 mM) and high plasma glucose levels (9 mM) using the primed constant -[3-³H]glucose infusion technique. In the presence of high glucose levels there was a dose-dependent increase in glucose elimination by CCK8. No effect of CCK8 on glucose production at a high glucose infusion rate (500 mg/kg per h) was observed in contrast to a low glucose infusion rate (100 mg/kg per h); plasma glucagon levels were elevated. All effects on glucose production and elimination were specific, since they were abolished by the CCK receptor antagonist L-364,718. In summary, glucose elimination was slightly increased by CCK8 at low glucose levels but increased to a greater extent at high glucose levels; glucose production was increased by CCK8 only at low glucose levels. Thus, CCK is a regulator of glucose homeostasis.     

High glucose concentrations inhibit glucose phosphorylation, but not glucose transport, in human endothelial cells.

Glucose uptake is autoregulated in a variety of cell types and it is thought that glucose transport is the major step that is subjected to control by sugar availability. Here, we examined the effect of high glucose concentrations on the rate of glucose uptake by human ECV-304 umbilical vein-derived endothelial cells. A rise in the glucose concentration in the medium led a dose-dependent decrease in the rate of 2-deoxyglucose uptake. The effect of high glucose was independent of protein synthesis and the time-course analysis indicated that it was relatively slow. The effect was not due to inhibition of glucose transport since neither the expression nor the subcellular distribution of the major glucose transporter GLUT1, nor the rate of 3-O-methylglucose uptake was affected. The total in vitro assayed hexokinase activity and the expression of hexokinase-I were similar in cells treated or not with high concentrations of glucose. In contrast, exposure of cells to a high glucose concentration caused a marked decrease in phosphorylated 2-deoxyglucose/free 2-deoxyglucose ratio. This suggests the existence of alterations in the rate of in vivo glucose phosphorylation in response to high glucose. In summary, we conclude that ECV304 human endothelial cells reduce glucose utilization in response to enhanced levels of glucose in the medium by inhibiting the rate of glucose phosphorylation, rather than by blocking glucose transport. This suggests a novel metabolic effect of high glucose on cellular glucose utilization.     

Endocytosis and Vacuolar Degradation of the Yeast Cell Surface Glucose Sensors Rgt2 and Snf3

Sensing and signaling the presence of extracellular glucose is crucial for the yeast Saccharomyces cerevisiae because of its fermentative metabolism, characterized by high glucose flux through glycolysis. The yeast senses glucose through the cell surface glucose sensors Rgt2 and Snf3, which serve as glucose receptors that generate the signal for induction of genes involved in glucose uptake and metabolism. Rgt2 and Snf3 detect high and low glucose concentrations, respectively, perhaps because of their different affinities for glucose. Here, we provide evidence that cell surface levels of glucose sensors are regulated by ubiquitination and degradation. The glucose sensors are removed from the plasma membrane through endocytosis and targeted to the vacuole for degradation upon glucose depletion. The turnover of the glucose sensors is inhibited in endocytosis defective mutants, and the sensor proteins with a mutation at their putative ubiquitin-acceptor lysine residues are resistant to degradation. Of note, the low affinity glucose sensor Rgt2 remains stable only in high glucose grown cells, and the high affinity glucose sensor Snf3 is stable only in cells grown in low glucose. In addition, constitutively active, signaling forms of glucose sensors do not undergo endocytosis, whereas signaling defective sensors are constitutively targeted for degradation, suggesting that the stability of the glucose sensors may be associated with their ability to sense glucose. Therefore, our findings demonstrate that the amount of glucose available dictates the cell surface levels of the glucose sensors and that the regulation of glucose sensors by glucose concentration may enable yeast cells to maintain glucose sensing activity at the cell surface over a wide range of glucose concentrations.     

Transport of glucose, gluconate, and methyl alpha-D-glucoside by Pseudomonas aeruginosa

Glucose transport by Pseudomonas aeruginosa was studied. These studies were enhanced by the use of a mutant, strain PAO 57, which was unable to grow on glucose but which formed the inducible glucose transport system when grown in media containing glucose or other inducers such as 2-deoxy-d-glucose. Both PAO 57 and parental strain PAO transported glucose with an apparent K(m) of 7 muM. Free glucose was concentrated intracellularly by P. aeruginosa PAO 57 over 200-fold above the external level. These data constitute direct evidence that glucose is transported via active transport by P. aeruginosa. Various experimental data clearly indicated that P. aeruginosa PAO transported methyl alpha-d-glucose (alpha-MeGlc) via the glucose transport system. The apparent K(m) of alpha-MeGlc transport was 7 mM which indicated a 1,000-fold lower affinity of the glucose transport system for alpha-MeGlc than for glucose. While only unchanged alpha-MeGlc was detected intracellularly in P. aeruginosa, alpha-MeGlc was actually concentrated intracellularly less than 2-fold over the external level. Membrane vesicles of P. aeruginosa PAO retained transport activity for gluconate. This solute was concentrated intravesicularly several-fold over the external level. A component of the glucose transport system is believed to have been lost during vesicle preparation since glucose per se was not transported. Instead; glucose was converted to gluconate by membrane-associated glucose dehydrogenase and gluconate was then transported into the vesicles. Although this may constitute an alternate system for glucose transport, it is not a necessary prerequisite for glucose transport by intact cells since P. aeruginosa PAO 57, which lacks glucose dehydrogenase, was able to transport glucose at a rate equal to the parental strain.     

Effects of hypo and hyperthyroidism on the response to glucose loading of blood glucose, ketones and insulin and liver glycogen as studied in the fasted rat

After receiving an i.p. glucose load, 24 h fasted thyroidectomized rats showed a progressive increase in blood glucose and a slow decrease in blood ketone bodies. Both liver glycogen and plasma insulin levels showed no differences within 60 min of the glucose administration. It is suggested that the glucose intolerance in these animals is partly due to an insulin deficiency. Thyroidectomized rats treated daily with 25 microgram of L-thyroxine/100 g body weight for 40 days responded to the glucose test with a supranormal and more persistent elevation of blood glucose but with a faster and a greater fall in blood ketone bodies, as compared to controls. Sixty min after the glucose loading, liver glucogen levels were lower and plasma insulin were slightly higher than controls. It is suggested that a diminished extraction of glucose during transhepatic passage can be responsible for the impaired glucose tolerance observed in the hyperthyroid animals.     

Determination of a glucose-dependent futile recycling rate constant from an intraperitoneal glucose tolerance test

Increased glucose cycling between glucose and glucose-6-phosphate is characteristic of insulin resistance and hyperglycemia seen with Type II diabetes. Traditionally, glucose cycling is determined by the difference between hepatic glucose output measured with separate [2-3H]glucose and [6-3H]glucose infusions. We demonstrate a novel method for determining hepatic glucose recycling from an intraperitoneal glucose tolerance test (IPGTT). A single tracer, [1, 2-13C(2)]glucose (a M2 glucose isotopomer), was administered at 1mg/g body weight to 4-month-old C57BL/6 mice. Hepatic glucose recycling was monitored by the appearance of a plasma M1 isotopomer of glucose, which is produced by the action of the pentose cycle on the M2 glucose isotopomer in the liver. The initial M2 enrichment was 56% and decreased to 13% at the end of 3 h, and the M1 enrichment peaked at 2 h. The ratio of plasma M1/M2 glucose increased linearly with time to approximately 25%, and the regression of the M1/M2 ratio against time gives a slope, termed the in vivo glucose-dependent futile recycling rate constant k(HR). k(HR) estimates glucose/glucose-6-phosphate futile cycling, along with glucose recycling through the pentose cycle. These observations demonstrate complex substrate cycling during an IPGTT using a single stable isotope tracer.     

Absorption of an oral glucose load in the dog

Absorption of glucose from the gut was estimated in trained unanesthetized dogs given a glucose load of 1-25 g (14C)glucose by stomach tube. The rate of absorption of glucose was calculated from the concentration and specific activity of glucose in the portal vein and in an "arterialized" peripheral vein. When the rate was integrated over time it was found that 94 +/- 4% of the administered glucose was recovered from the portal vein as glucose; this was unrelated to the size of the glucose load. It is concluded that absorption does not entail a significant loss or conversion to glucose metabolites.     

Glucose regulates glucokinase activity in cultured islets from rat pancreas

In this study, we have used isolated pancreatic islets cultured for 7 days in 3 or 30 mM glucose to explore whether glucokinase is induced or activated by high glucose concentrations and has related enzyme activity to glucose-stimulated insulin release. Islets cultured in low glucose medium or low glucose medium plus 350 ng/ml insulin did not respond to high glucose stimulation. Islets cultured in medium containing high glucose concentrations showed a high rate of basal insulin secretion when perifused with 5 mM glucose, and the insulin release was greatly augmented in a biphasic secretion profile when the glucose concentration was raised to 16 mM. Islet glucokinase and hexokinase activities were determined by a sensitive and specific fluorometric method. Glucokinase activity was reduced to approximately 50% in islets cultured in low glucose medium with or without insulin present compared to results with fresh islets. However, islets cultured in 30 mM glucose showed that glucokinase activity was elevated to 236% compared to results with fresh islets. It is concluded that (a) glucose is the physiological regulator of glucokinase in the islet of Langerhans and that (b) the activity of glucokinase plays a crucial role in glucose-induced insulin secretion.     

A study of the Crabtree effect in Novikoff ascites-hepatoma cells

Novikoff ascites-hepatoma cells show no Crabtree effect on the addition of glucose to tumour-cell suspensions, and convert a significant part of the added glucose into glycogen. Treatment of the cells with 2-deoxyglucose or glucose in the presence of iodoacetate inhibits respiration and decreases glycogen synthesis from glucose. Short-term experiments indicate a slight inhibition of glucose uptake for a brief period, due either to ATP accumulation in the mitochondria or to glucose 6-phosphate-mediated inhibition of hexokinase. Utilization of glucose metabolites and ATP for glycogen synthesis appears to remove inhibition of glucose uptake, and perhaps accounts for the absence of respiratory inhibition, by relieving a deficiency of ADP for the mitochondria. Decreased respiration in the presence of 2-deoxyglucose or glucose in the presence of iodoacetate could be due to the change in mitochondrial structure or permeability, caused by the significant loss of adenine nucleotides.     

Isolation and characterization of Chinese hamster ovary cell mutants defective in glucose transport

Cultured Chinese hamster ovary (CHO) cells possess an insulin-sensitive facilitated diffusion system for glucose transport. Mutant clones of CHO cells defective in glucose transport were obtained by repeating the selection procedure, which involved mutagenesis with ethyl methanesulfonate, radiation suicide with tritiated 2-deoxy-D-glucose, the polyester replica technique and in situ autoradiographic assaying for glucose accumulation. On the first selection, we obtained mutants exhibiting about half the glucose uptake activity of parental CHO-K1 cells and half the amount of a glucose transporter, the amount of which was determined by immunoblotting with an antibody to the human erythrocyte glucose transporter. The second selection, starting from one of the mutants obtained in the first-step selection, yielded a strain, GTS-31, in which both glucose uptake activity and the quantity of the glucose transporter were 10-20% of the levels in CHO-K1 cells, whereas the responsiveness of glucose transport to insulin, and the activities of leucine uptake and several glycolytic enzymes remained unchanged. GTS-31 cells grew slower than CHO-K1 cells at both 33 and 40 degrees C, and in a medium containing a low concentration of glucose (0.1 mM), the mutant cells lost the ability to form colonies. All the three spontaneous GTS-31 cell revertants, which were isolated by growing the mutant cells in medium containing 0.1 mM glucose, exhibited about half the glucose uptake activity and about half the amount of glucose transporter, as compared to in CHO-K1 cells, these characteristics being similar to those of the first-step mutant. These results indicate that the decrease in glucose uptake activity in strain GTS-31 is due to a mutation which induces a reduction in the amount of the glucose transporter, providing genetic evidence that the glucose transporter functions as a major route for glucose entry into CHO-K1 cells.     

The rate-limiting step for glucose transport into the hypothalamus is across the blood–hypothalamus interface

Specialized glucosensing neurons are present in the hypothalamus, some of which neighbor the median eminence, where the blood–brain barrier has been reported leaky. A leaky blood–brain barrier implies high tissue glucose levels and obviates a role for endothelial glucose transporters in the control of hypothalamic glucose concentration, important in understanding the mechanisms of glucose sensing We therefore addressed the question of blood–brain barrier integrity at the hypothalamus for glucose transport by examining the brain tissue-to-plasma glucose ratio in the hypothalamus relative to other brain regions. We also examined glycogenolysis in hypothalamus because its occurrence is unlikely in the potential absence of a hypothalamus–blood interface. Across all regions the concentration of glucose was comparable at a given plasma glucose concentration and was a near linear function of plasma glucose. At steady-state, hypothalamic glucose concentration was similar to the extracellular hypothalamic glucose concentration reported by others. Hypothalamic glycogen fell at a rate of ∼1.5 μmol/g/h and remained present in substantial amounts. We conclude for the hypothalamus, a putative primary site of brain glucose sensing that: the rate-limiting step for glucose transport into brain cells is at the blood–hypothalamus interface, and that glycogenolysis is consistent with a substantial blood -to- intracellular glucose concentration gradient.     

High frequency of glucose-utilizing mutants in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 has conventionally been considered unable to use glucose as a carbon substrate for growth. The genome sequence of S. oneidensis MR-1 however suggests the ability to use glucose. Here, we demonstrate that during initial glucose exposure, S. oneidensis MR-1 quickly and frequently gains the ability to utilize glucose as a sole carbon source, in contrast to wild-type S. oneidensis, which cannot immediately use glucose as a sole carbon substrate. High-performance liquid chromatography and (14)C glucose tracer studies confirm the disappearance in cultures and assimilation and respiration, respectively, of glucose. The relatively short time frame with which S. oneidensis MR-1 gained the ability to use glucose raises interesting ecological implications.     

Novel FAD-dependent glucose dehydrogenase for a dioxygen-insensitive glucose biosensor

A novel FAD-dependent glucose dehydrogenase (FAD-GDH) was found and its enzymatic property for glucose sensing was characterized. FAD-GDH oxidized glucose in the presence of some artificial electron acceptors, except for O2, and exhibited thermostability, high substrate specificity and a large Michaelis constant for glucose. FAD-GDH was applied to an amperometric glucose sensor with Fe(CN)6(3-) as a soluble mediator. The use of a relatively high concentration of Fe(CN)6(3-) resulted in a good linearity between the current response and the glucose concentration, taking into account a large Michaelis constant for Fe(CN)6(3-). The glucose sensor was completely insensitive to O2 and responded linearly to glucose up to 30 mM. Compared to glucose, the response to other saccharides was negligible. The sensor can be stored at room temperature in a desiccator for at least one month without any change in the response or activity.     

Blood-Brain Glucose Transfer in Spreading Depression

Abstract Spreading depression in rat brain cortex is associated with a twofold increase of cerebral blood flow. It is not known whether this increase is coupled to increases of cerebral metabolic rate and glucose transport from blood to brain. During the passage of a single spreading depression, we measured blood-brain glucose transport and glucose metabolism in rat cerebral cortex by single intravenous injection of tracer glucose. Blood flow and tissue content of glucose were measured as well. Reduction of tissue glucose and the consequent increase of net transfer of glucose from blood to brain were consistent with a threefold increase of the consumption of glucose before the increase of blood flow. There was no increase of unidirectional blood-brain transfer.