Carbohydrate metabolism in pregnancy. Part II. Relation between maternal glucose tolerance and glucose metabolism in the newborn.

The objective of clinical management of the pregnant diabetic woman is to prevent the serious adverse effects of an abnormal glucose environment on the fetus. Neonatal glucose assimilation and insulin release over the first two hours of life were correlated with various indices of maternal carbohydrate metabolism in the third trimester. Of the 31 mothers studied 21 were defined as normal and 10 as having chemical diabetes. Neontal glucose assimilation during the first two hours of life correlated strongly with functions of both maternal glucose tolerance and mean diurnal glucose level, the strongest correlation being with the area under the three-hour oral glucose tolerance curve (P less than 0.001), Two-hour neonatal plasma glucose values of under 1.7 mmol/1 (30 mg/100 ml) were found only in the newborn of women whose glucose tolerance area measured over 41.6 area units (750 traditional units); thus, even in the borderline diabetic range glucose tolerance testing during the last trimester of pregnancy may be valuable in predicting likelihood of neonatal hypoglycaemia. The findings also shed light on the possible sensitizing role of mild maternal hyperglycaemia on fetal insulin production and secretion.     

Insulin-stimulated glucose uptake and fasting blood glucose.

Changes in insulin-stimulated glucose metabolism were studied in young and aged subjects, subjects with impaired glucose tolerance, and patients with NIDDM by means of the glucose clamp technique. The diabetic group includes obese and non-obese patients treated without insulin and non-obese patients treated with insulin. The glucose disposal rate (GDR) was decreased in aged subjects (5.8 +/- 0.4 mg/kg/min) compared with young controls (7.4 +/- 0.3 mg/kg/min). In patients with IGT, it was further decreased to 3.6 +/- 0.5 mg/kg/min, which was comparable to the rate in NIDDM without insulin treatment (3.3 +/- 0.4 mg/kg/min). There were no differences in the GDR between obese (3.0 +/- 0.3 mg/kg/min) and non-obese (3.4 +/- 0.6 mg/kg/min) diabetic patients. In insulin-treated diabetic patients, GDR ranged widely, but the mean value was partially normalized (5.2 +/- 0.9 mg/kg/min). In the diabetic group, no correlation was observed between fasting blood glucose and GDR. These results suggest that in the course of developing NIDDM, a decrease in insulin-stimulated glucose uptake precedes a rise in fasting blood glucose. Thus, as previously reported for Caucasian NIDDM patients, resistance to insulin-stimulated glucose uptake may be one of the basic defects in Japanese patients with NIDDM. The degree of glycemia, however, is not directly related to the magnitude of the defect in insulin action.     

The role of glucose limitation in the regulation of the transport of glucose, gluconate and 2-oxogluconate, and of glucose metabolism in Pseudomonas aeruginosa.

The pathway of glucose metabolism in Pseudomonas aeruginosa was regulated by the availability of glucose and related compounds. On changing from an ammonium limitation to a glucose limitation, the organism responded by adjusting its metabolism substantially from the extracellular direct oxidative pathway to the intracellular phosphorylative route. This change was achieved by repression of the transport systems for gluconate and 2-oxogluconate and of the associated enzymes for 2-oxogluconate metabolism and gluconate kinase, while increasing the levels of glucose transport, hexokinase and glucose 6-phosphate dehydrogenase. The role of gluconate, produced by the action of glucose dehydrogenase, as a major inhibitory factor for glucose transport, and the possible significance of these regulatory mechanisms to the organism in its natural environment, are discussed.     

Effects of insulin on glucose transport and glucose transporters in rat heart.

The effect of insulin on glucose transport and glucose transporters was studied in perfused rat heart. Glucose transport was measured by the efflux of labelled 3-O-methylglucose from hearts preloaded with this hexose. Insulin stimulated 3-O-methylglucose transport by: (a) doubling the maximal velocity (Vmax); (b) decreasing the Kd from 6.9 to 2.7 mM; (c) increasing the Hill coefficient toward 3-O-methylglucose from 1.9 to 3.1; (d) increasing the efficiency of the transport process (k constant). Glucose transporters in enriched plasma and microsomal membranes from heart were quantified by the [3H]cytochalasin-B-binding assay. When added to normal hearts, insulin produced the following changes in the glucose transporters: (a) it increased the translocation of transporters from an intracellular pool to the plasma membranes; (b) it increased (from 1.6 to 2.7) the Hill coefficient of the transporters translocated into the plasma membranes toward cytochalasin B, suggesting the existence of a positive co-operativity among the transporters appearing in these membranes; (c) it increased the affinity of the transporters (and hence, possibly, of glucose) for cytochalasin B. The data provide evidence that the stimulatory effect of insulin on glucose transport may be due not to the sole translocation of intracellular glucose transporters to the plasma membrane, but to changes in the functional properties thereof.     

Continuous glucose monitoring in cystic fibrosis patients according to the glucose tolerance.

Cystic fibrosis (CF) is associated with a long preclinical state of abnormal glucose tolerance. The aim of this study was (i) to evaluate the profile of glucose tolerance in young adults with CF and (ii) to compare these results with those obtained by a continuous subcutaneous glucose monitoring (CGMS). CF subjects with fasting glycemia inferior to 126 mg/dl were included in the study. An oral glucose tolerance test (OGTT) identified the subjects either with a normal glucose tolerance (NGT), or impaired glucose tolerance (IGT), or diabetes. CGMS (Medtronic) was performed during 3 days to analyze mean glucose level, high glucose excursions, and glucose area under the curve (AUC). Forty-nine patients were included in the study. NGT (n=22), IGT (n=17), and diabetes groups (n=10) were comparable except with regard to age and BMI (p<0.001). HbA1c values in diabetes group were significantly higher (p<0.001) than in NGT and IGT groups. CGMS revealed peaks of glucose values superior to 200 mg/dl at least once after a meal in 8 patients (36%) with NGT, in 9 patients (52%) with IGT, and in all patients with diabetes (p<0.01). Mean CGMS glucose and glucose AUC values increased in patients with diabetes compared to patients with NGT and IGT (p<0.05). Peak of CGMS glucose reached 182+/-60 mg/dl in NGT group despite the normal glucose profile at OGTT. In conclusion, CGMS revealed pathological glucose excursions not only in patients with impaired glucose tolerance at OGTT but also in patients with a normal glycemic profile. CGMS could be a useful tool for the early detection of hyperglycemia in patients with CF.     

Characterization of transmembrane movement of glucose and glucose analogs in Streptococcus mutants Ingbritt.

The transmembrane movement of radiolabeled, nonmetabolizable glucose analogs in Streptococcus mutants Ingbritt was studied under conditions of differing transmembrane electrochemical potentials (delta psi) and pH gradients (delta pH). The delta pH and delta psi were determined from the transmembrane equilibration of radiolabeled benzoate and tetraphenylphosphonium ions, respectively. Growth conditions of S. mutants Ingbritt were chosen so that the cells had a low apparent phosphoenolpyruvate (PEP)-dependent glucose:phosphotransferase activity. Cells energized under different conditions produced transmembrane proton potentials ranging from -49 to -103 mV but did not accumulate 6-deoxyglucose intracellularly. An artificial transmembrane proton potential was generated in deenergized cells by creating a delta psi with a valinomycin-induced K+ diffusion potential and a delta pH by rapid acidification of the medium. Artificial transmembrane proton potentials up to -83 mV, although producing proton influx, could not accumulate 6-deoxyglucose in deenergized cells or 2-deoxyglucose or thiomethylgalactoside in deenergized, PEP-depleted cells. The transmembrane diffusion of glucose in PEP-depleted, KF-treated cells did not exhibit saturation kinetics or competitive inhibition by 6-deoxyglucose or 2-deoxyglucose, indicating that diffusion was not facilitated by a membrane carrier. As proton-linked membrane carriers have been shown to facilitate diffusion in the absence of a transmembrane proton potential, the results therefore are not consistent with a proton-linked glucose carrier in S. mutans Ingbritt. This together with the lack of proton-linked transport of the glucose analogs suggests that glucose transmembrane movement in S. mutans Ingbritt is not linked to the transmembrane proton potential.     

New lessons in the regulation of glucose metabolism taught by the glucose 6-phosphatase system.

The operation of glucose 6-phosphatase (EC 3.1.3.9) (Glc6Pase) stems from the interaction of at least two highly hydrophobic proteins embedded in the ER membrane, a heavily glycosylated catalytic subunit of m 36 kDa (P36) and a 46-kDa putative glucose 6-phosphate (Glc6P) translocase (P46). Topology studies of P36 and P46 predict, respectively, nine and ten transmembrane domains with the N-terminal end of P36 oriented towards the lumen of the ER and both termini of P46 oriented towards the cytoplasm. P36 gene expression is increased by glucose, fructose 2,6-bisphosphate (Fru-2,6-P2) and free fatty acids, as well as by glucocorticoids and cyclic AMP; the latter are counteracted by insulin. P46 gene expression is affected by glucose, insulin and cyclic AMP in a manner similar to P36. Accordingly, several response elements for glucocorticoids, cyclic AMP and insulin regulated by hepatocyte nuclear factors were found in the Glc6Pase promoter. Mutations in P36 and P46 lead to glycogen storage disease (GSD) type-1a and type-1 non a (formerly 1b and 1c), respectively. Adenovirus-mediated overexpression of P36 in hepatocytes and in vivo impairs glycogen metabolism and glycolysis and increases glucose production; P36 overexpression in INS-1 cells results in decreased glycolysis and glucose-induced insulin secretion. The nature of the interaction between P36 and P46 in controling Glc6Pase activity remains to be defined. The latter might also have functions other than Glc6P transport that are related to Glc6P metabolism.     

Tryptophan and the control of plasma glucose concentrations in the rat.

1. Injection of L-tryptophan (750 mg/kg body wt.) led to pronounced hypoglycaemia in fed and 48 h-starved rats. 2. The hypoglycaemic effect is blocked by pretreament with p-chlorophenylalanine, compound MK-486 [Carbidopa: L-alpha-(3,4-dihydroxybenzyl)-alpha-hydrazinopropionic acid monohydrate] or methysergide, and potentiated by pargyline. 3. 5-Hydroxy-L-tryptophan is more potent and induces a more rapid hypoglycaemia than does tryptophan. Other tryptophan metabolites were not associated with hypoglycaemia. 4. Adrenalectomy increases, and acute experimental diabetes strongly decreases, the sensitivity of rats to tryptophan induction of hypoglycaemia. Diabetic animals were also insensitive to 5-hydroxytryptophan. 5. Metabolite concentration changes in the livers from tryptophan-treated 48h-starved and diabetic animals were consistent with a rapid inhibition of gluconeogenesis. This did not correlate with the hypoglycaemic response. 6. Tryptophan treatment was associated with a significant increase in the plasma [beta-hydroxybutyrate]/[acetoacetate] ratio; there were no changes in the plasma concentrations of urea, triacyglycerol, non-esterified fatty acids and glycerol. 7. These observations suggest that the hypoglycaemic action of tryptophan is mediated through formation of intracellular 5-hydroxytryptamine, and is unrelated to the inhibition of gluconeogenesis. It is unlikely that this increased synthesis of 5-hydroxytryptamine involves directly either the adrenal glands or the central nervous system.     

First-pass hepatic uptake and utilization of glucose in the rat.

The concentration of NADH was determined a high-oxidative muscle (soleus) and a high-glycolytic muscle (extensor digitorum longus, EDL) from resting rats. The NADH content of freeze-clamped control muscles was 0.35 +/- 0.04 (mean +/- S.D.) and 0.31 +/- 0.04 mmol/kg dry wt. in EDL and soleus respectively, and increased to peak values of 0.58 +/- 0.05 (EDL) and 0.87 +/- 0.10 (soleus) after 10 min of NaCN treatment. The [lactate]/[pyruvate] ratio, which was not significantly changed in soleus and increased only slightly in EDL after NaCN incubation, shows that only minor changes occurred in the cytosolic NADH concentration. Provided that the major part of muscle NADH is located in the mitochondria it can be calculated that the mitochondrial NADH content in skeletal muscle at rest is about 36 (soleus) and 60% (EDL) of the anoxic value, respectively. These results are in contrast with previous studies with the surface-fluorescence technique, where mitochondrial NAD appeared to be almost completely reduced in resting skeletal muscle.     

Insulin and glucose in neonatal calves after peroral insulin and intravenous glucose administration.

Effects of peroral insulin on plasma concentrations of immunoreactive insulin (IRI) and glucose in newborn calves were studied. Bovine insulin was administered in amounts of 0.5 mg/kg body weight immediately preceding first colostrum. Thereafter, neither an increased IRI response nor a lowering of blood glucose level were observed, indicating that insulin was either not absorbed from the intestine or possibly retained in the liver. Feeding of whole milk was followed by a higher rise in IRI and glucose concentrations than feeding of colostrum after po insulin. However, when compared with 1-3-month old calves, IRI responses to feeding and to iv infused glucose were markedly smaller on the first and second day of life respectively, while glucose responses were similar. This indicates that insulin secretory mechanisms are not fully developed in the newborn calf.     

Insulin and the way the brain handles glucose.

—In living rats the concentration of insulin in the circulating blood was raised and independently of this the glucose concentration in the blood plasma was varied from hyperglycaemic to hypoglycaemic levels. Hyperglycaemia increased the influx of glucose into the brain and it also, for a limited period, increased the glucose gain by the brain. Insulin, on the other hand, did not affect influx but significantly increased the gain of glucose by the brain. It is suggested that although both hyperglycaemia and insulin can increase glucose gain by the brain they do so in entirely different ways.     

The fate of labelled glucose molecules in the rat adipocyte. Dependence on glucose concentration

Isolated rat adipocytes were incubated with 15 nM [3-3H]glucose or 100 nM [U-14C]glucose with or without insulin and in the absence or presence of unlabelled glucose. Following a 2 h incubation with 15 nM [3-3H]glucose, about two thirds of the cell-associated 3H-labelled metabolic products were hydrophilic largely anionic intermediates and about one third was lipids. The equivalent values were 40 and 60%, respectively, when using 100 nM [U-14C]glucose. The only 14C-labelled metabolite escaping to the incubation medium was 14CO2, which accounted for about 15% of the rate of metabolism. Therefore, the rate of incorporation of 100 nM [U-14C]glucose into the cell-associated metabolites was quite a good measure of its net influx rate. The conversion of the two tracers to the sum of the metabolic products in cells treated with a maximally stimulating insulin concentration remained constant with glucose concentrations up to about 100 microM and then decreased progressively. The incorporation of radioactivity into the different metabolites varied markedly over the glucose concentration range 0-100 microM, presumably due to the saturation of different metabolic pools at different glucose concentrations. This variation was much less in cells not stimulated with insulin. Consequently, the maximal effect of insulin on the incorporation of the tracers into a given metabolite (e.g., labelled lipids) varied over the entire glucose concentration range. In addition, the apparent sensitivity (ED50) with respect to the incorporation into a given metabolite was also dependent on the glucose concentration.     

Transport enhancement and reversal: glucose and 3-O-methyl glucose.

In chick embryo fibroblast cultures the 15- to 30-fold enhancement of D-glucose uptake observed when cells are starved of glucose for 24 hours is not duplicated for derivatives of glucose that compete effectively for uptake and have generally been considered to use the same carrier. 2-deoxy-D-glucose, D-mannose, D-galactose and D-glucosamine are derepressed progressively less sharply in that order with glucosamine uptake never more than doubled by starvation. D-glucose at a concentration of 5.5 mM in the 24-hour conditioning medium is a strong "repressor" resulting in low "transport" behavior for each of the five sugars cited. D-glucosamine is equally effective at the same concentration. A 10-fold reduction in the concentration of glucosamine (0.55 mM) allows for the escape from repression of mannose, glucose, and deoxyglucose uptake while the others remain repressed. Mannose uptake escapes as well when the glucose concentration in the "conditioning" medium is similarly reduced. Under certain conditions of starvation and cell density dramatic effects of supplemental stimulation by insulin can be achieved. Insulin withdrawal interrupts the supplemental stimulation process. Cycloheximide, actinomycin D and cordycepin block both non-insulin and insulin-induced derepression. Short exposure (15-30 minutes) of 24-hour starved cells to glucose (5.5 mM) reduces glucose sharply but does not affect 3-O-methyl glucose uptake. If the exposure is to 2-deoxyglucose (5.5 mM) further derepression of glucose uptake results.     

Antagonistic regulation of the glucose/glucose 6-phosphate cycle by insulin and glucagon in cultured hepatocytes.

Flux through the glucose/glucose 6-phosphate cycle in cultured hepatocytes was measured with radiochemical techniques. Utilization of [2-3H]glucose was taken as a measure of glucokinase flux. Liberation of [14C]glucose from [U-14C]glycogen and from [U-14C]lactate, as well as the difference between the utilization of [2-3H]glucose and of [U-14C]glucose, were taken as measures of glucose-6-phosphatase flux. At constant 5 mM-glucose and 2 mM-lactate concentrations insulin increased glucokinase flux by 35%; it decreased glucose-6-phosphatase flux from glycogen by 50%, from lactate by 15% and reverse flux from external glucose by 65%, i.e. overall by 40%. Glucagon had essentially no effect on glucokinase flux; it enhanced glucose-6-phosphatase flux from glycogen by 700%, from lactate by 45% and reverse flux from external glucose by 20%, i.e. overall by 110%. At constant glucose concentrations cellular glucose 6-phosphate concentrations were essentially not altered by insulin, but were increased by glucagon by 230%. In conclusion, under basic conditions without added hormones the glucose/glucose 6-phosphate cycle showed only a minor net glucose uptake, of 0.03 mumol/min per g of hepatocytes; this flux was increased by insulin to a net glucose uptake of 0.21 mumol/min per g and reversed by glucagon to a net glucose release of 0.22 mumol/min per g. Since the glucose 6-phosphate concentrations after hormone treatment did not correlate with the glucose-6-phosphatase flux, it is suggested that the hormones influenced the enzyme activity directly.     

Control of glucose metabolism in isolated acini of the lactating mamary gland of the rat. Effects of oleate on glucose utilization and lipogenesis.

Oleate (1mM) had only small inhibitory effects on glucose utilization and lipogenesis in acini isolated from rat mammary gland. Esterification of [1-14C]oleate was unaffected by insulin but were decreased by 60% by acetoacetate (2mM). Glycerol (1mM), but not insulin, relieved this inhibition. These experiments provide further support for the role of acetoacetate in regulating substrate utilization by the gland.     

Evaluating in vitro and in vivo the interference of ascorbate and acetaminophen on glucose detection by a needle-type glucose sensor.

The aim of this work was to assess, in vitro and in vivo, the interference of ascorbate and acetaminophen on glucose measurements by a needle-type glucose sensor detecting hydrogen peroxide generated during the enzymatic oxidation of glucose, and to ascertain whether the protection against interference by the membranes used in the construction of the electrode is feasible. The oxidation of ascorbate and acetaminophen on a platinum electrode set at a 650 mV potential yielded a current representing 75 +/- 5% and 25 +/- 6% of that generated by the oxidation of an equimolar concentration of hydrogen peroxide, respectively. The bias introduced by the presence of 100 mumol l-1 ascorbate on the reading of 5 mmol l-1 glucose by the complete sensor (electrode + membranes) would be minimal (approximately 0.4 mmol l-1). By contrast, the bias introduced by 200 mumol l-1 of acetaminophen (a plasma concentration easily reached in clinical practice) was about 7 mmol l-1. The sensor was implanted subcutaneously in anaesthetized rats (n = 3). Using the current generated in the presence of a plasma acetaminophen concentration of about 200 mumol l-1 for glucose monitoring would lead to a major underestimation (approx. 6 mmol l-1) of subcutaneous glucose concentrations.