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A Burchell

Publications and source records attributed to A Burchell.

At least 37 records · Page 2Linked to original sources
PubMed1998

Heterogeneity of glucose transport in rat liver microsomal vesicles.

Glucose transport across the membrane of rat liver microsomal vesicles was studied by a rapid filtration method in three different experimental systems: (i) inward transport in the presence of extravesicular glucose, (ii) efflux from passively preloaded vesicles, and (iii) efflux of glucose generated intravesicularly by glucose-6-phosphatase upon addition of glucose 6-phosphate were investigated. The apparent intravesicular glucose space estimated with the rapid filtration method was lower than the total microsomal glucose accessible space both the in the steady-state phase of uptake and at the starting point of efflux: 0.5 versus 2.3 microl/mg protein. The initial rate of influx/efflux was dependent on the extravesicular/intravesicular glucose concentration and was much lower than the rate of influx estimated previously by the light-scattering technique. Both influx and efflux could be inhibited by N-ethylmaleimide and possibly became saturable at high (>100 mM) glucose concentration. Known inhibitors of GLUT transporters (genistein, cytochalasin B, phloretin, and hexoses) did not affect glucose influx. The time course of glucose efflux from vesicles preincubated in the presence of glucose 6-phosphate was similar to that from glucose-loaded vesicles. These data together with that obtained previously (by a light-scattering technique; Marcolongo, P., Fulceri, R., Giunti, R., Burchell, A., and Benedetti, A. (1996) Biochem. Biophys. Res. Commun. 219, 916-922) indicate that microsomal vesicles are heterogeneous regarding their glucose-transporting properties and that glucose transport is bidirectional and its feature meets the requirements of a facilitative transport.

Animals↗
PubMed1998

Structure and mutation analysis of the glycogen storage disease type 1b gene.

Glycogen storage disease (GSD) 1b is the deficiency of endoplasmic reticulum glucose-6-phosphate (G6P) transport. We here report the structure of the gene encoding a protein likely to be responsible for G6P transport, and its mapping to human chromosome 11q23.3. The gene is composed of nine exons spanning a genomic region of approximately 4 kb. Primers based on the genomic sequence were used in single strand conformation polymorphism (SSCP) analysis and mutations were found in six out of seven GSD 1b patients analysed.

Antiporters↗
PubMed1998

A different isoform of the transport protein mutated in the glycogen storage disease 1b is expressed in brain.

There are differences in the kinetic properties of the liver and brain microsomal glucose-6-phosphate transport systems suggesting the possibility of tissue specific isoforms. The availability of a human liver cDNA sequence which is mutated in patients with deficiencies of liver microsomal glucose-6-phosphate transport (glycogen storage disease 1b) made it possible to determine if a brain isoform exists. Northern blots of liver and brain RNA revealed that the mRNA of the brain form is slightly longer than the liver one. Isolation and sequencing of the respective human brain cDNA revealed that the brain protein has an additional 22 amino acid sequence.

Amino Acid Sequence↗
PubMed1998

Three thiol groups are important for the activity of the liver microsomal glucose-6-phosphatase system. Unusual behavior of one thiol located in the glucose-6-phosphate translocase.

Liver microsomal glucose-6-phosphatase (Glc-6-Pase) is a multicomponent system involving both substrate and product carriers and a catalytic subunit. We have investigated the inhibitory effect of N-ethylmaleimide (NEM), a rather specific sulfhydryl reagent, on rat liver Glc-6-Pase activity. Three thiol groups are important for Glc-6-Pase system activity. Two of them are located in the glucose-6-phosphate (Glc-6-P) translocase, and one is located in the catalytic subunit. The other transporters (phosphate and glucose) are not affected by NEM treatment. The NEM alkylation of the catalytic subunit sulfhydryl residue is prevented by preincubating the disrupted microsomes with saturating concentrations of substrate or product. This suggests either that the modified cysteine is located in the protein active site or that substrate binding hides the thiol group via a conformational change in the enzyme structure. Two other thiols important for the Glc-6-Pase system activity are located in the Glc-6-P translocase and are more reactive than the one located in the catalytic subunit. The study of the NEM inhibition of the translocase has provided evidence of the existence of two distinct areas in the protein that can behave independently, with conformational changes occurring during Glc-6-P binding to the transporter. The recent cloning of a human putative Glc-6-P carrier exhibiting homologies with bacterial phosphoester transporters, such as Escherichia coli UhpT (a Glc-6-P translocase), is compatible with the fact that two cysteine residues are important for the bacterial Glc-6-P transport.

Animals↗
PubMed1998

Glucocorticoid exposure in late gestation permanently programs rat hepatic phosphoenolpyruvate carboxykinase and glucocorticoid receptor expression and causes glucose intolerance in adult offspring.

Low birth weight in humans is predictive of insulin resistance and diabetes in adult life. The molecular mechanisms underlying this link are unknown but fetal exposure to excess glucocorticoids has been implicated. The fetus is normally protected from the higher maternal levels of glucocorticoids by feto-placental 11beta-hydroxysteroid dehydrogenase type-2 (11beta-HSD2) which inactivates glucocorticoids. We have shown previously that inhibiting 11beta-HSD2 throughout pregnancy in rats reduces birth weight and causes hyperglycemia in the adult offspring. We now show that dexamethasone (a poor substrate for 11beta-HSD2) administered to pregnant rats selectively in the last week of pregnancy reduces birth weight by 10% (P < 0.05), and produces adult fasting hyperglycemia (treated 5.3+/-0.3; control 4.3+/-0.2 mmol/ liter, P = 0.04), reactive hyperglycemia (treated 8.7+/-0.4; control 7.5+/-0.2 mmol/liter, P = 0.03), and hyperinsulinemia (treated 6.1+/-0.4; control 3.8+/-0.5 ng/ml, P = 0.01) on oral glucose loading. In the adult offspring of rats exposed to dexamethasone in late pregnancy, hepatic expression of glucocorticoid receptor (GR) mRNA and phosphoenolpyruvate carboxykinase (PEPCK) mRNA (and activity) are increased by 25% (P = 0.01) and 60% (P < 0.01), respectively, while other liver enzymes (glucose-6-phosphatase, glucokinase, and 11beta-hydroxysteroid dehydrogenase type-1) are unaltered. In contrast dexamethasone, when given in the first or second week of gestation, has no effect on offspring insulin/glucose responses or hepatic PEPCK and GR expression. The increased hepatic GR expression may be crucial, since rats exposed to dexamethasone in utero showed potentiated glucose responses to exogenous corticosterone. These observations suggest that excessive glucocorticoid exposure late in pregnancy predisposes the offspring to glucose intolerance in adulthood. Programmed hepatic PEPCK overexpression, perhaps mediated by increased GR, may promote this process by increasing gluconeogenesis.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1998

Microsomal steroid sulfatase: interactions with cytosolic steroid sulfotransferases.

Net sulfation of 4-methylumbelliferone in intact hepatocytes is regulated, in part, by substrate cycling between sulfotransferases (SULT) and arylsulfatases (ARS). Thus, ARS have the potential to influence rates of net sulfate conjugation of a variety of compounds in intact cells via interaction with SULT. Unlike ARSA and ARSB, which are lysosomal, steroid sulfate sulfatase (ARSC, also known as STS) is localized exclusively in the endoplasmic reticulum (ER). The present study was designed to assess the existence and extent of substrate cycling between steroids and their sulfate conjugates through ARSC and SULT, and also to initiate studies of the topology of the catalytic site of ARSC in the rat liver ER. Addition of rat liver microsomes to cytosol and 3'-phosphoadenosine 5'-phosphosulfate (PAPS) reduced rates of sulfation of dehydroepiandrosterone (DHEA) by SULT, and similarly hydrolysis of DHEA sulfate (DHEAS) was reduced when recombinant human hydroxysteroid SULT was added to rat liver microsomes in the presence of PAPS. There was no evidence for ARSC latency in the presence of detergent at either 4 or 37 degrees C, indicating that facilitated transport of steroid sulfates across the ER membrane may not be required for ARSC activity. The effect of proteases on ARSC activity in intact and disrupted microsomes was determined and compared with effects on components of the glucose-6-phosphatase system known to be localized on the lumenal and cytoplasmic surfaces of the ER. In contrast to the components of the glucose-6-phosphatase system, activity of ARSC in both intact and disrupted microsomes was substantially more resistant to protease inactivation. Our results indicate that substrate cycling of steroids and their sulfates does occur, and suggest that the active site of ARSC may be located within the ER membrane.

Animals↗
PubMed1998

Glycogen storage diseases and the liver.

Carbohydrate metabolism in the liver is responsible for plasma glucose homeostasis. Liver glycogen storage diseases are metabolic disorders which result in abnormal storage amounts and/or forms of glycogen, and often (but not always) have hepatomegaly and hypoglycaemia as presenting features. To understand the clinical complexity of the glycogen storage diseases, it is necessary to understand the properties and regulation of the proteins involved in glycogen metabolism. Advances in treatment have greatly improved metabolic control and hence the quality of life and survival. However, the lack of understanding of the molecular basis of some of the clinical features of glycogen storage diseases makes it difficult logically to devise optimal treatment regimens to prevent some of the long-term complications. Recently, molecular biology has greatly advanced our understanding of the proteins and genes involved in liver glycogen metabolism and has led to better and less invasive methods of diagnosis of these disorders.

Glycogen Storage Disease↗
PubMed1998

Liver microsomal transport of glucose-6-phosphate, glucose, and phosphate in type 1 glycogen storage disease.

The transport of glucose-6-phosphate (G6P), glucose, and orthophosphate into liver microsomes, isolated from six patients with various subtypes of type 1 glycogen storage disease (GSD), was measured using a light-scattering method. We found that G6P, glucose, and phosphate could all cross the microsomal membrane, in four cases of type 1a GSD. In contrast, liver microsomal transport of G6P and phosphate was deficient in the GSD 1b and 1c patients, respectively. These results support the involvement of multiple proteins (and genes) in GSD type 1. The results obtained with the light-scattering method are in accordance with conventional kinetic analysis of the microsomal glucose-6-phosphatase system. Therefore, this technique could be used to directly diagnose type 1b and 1c GSD.

Adult↗
PubMed1997

11beta-hydroxysteroid dehydrogenase type 1 knockout mice show attenuated glucocorticoid-inducible responses and resist hyperglycemia on obesity or stress.

Glucocorticoid hormones, acting via nuclear receptors, regulate many metabolic processes, including hepatic gluconeogenesis. It recently has been recognized that intracellular glucocorticoid concentrations are determined not only by plasma hormone levels, but also by intracellular 11beta-hydroxysteroid dehydrogenases (11beta-HSDs), which interconvert active corticosterone (cortisol in humans) and inert 11-dehydrocorticosterone (cortisone in humans). 11beta-HSD type 2, a dehydrogenase, thus excludes glucocorticoids from otherwise nonselective mineralocorticoid receptors in the kidney. Recent data suggest the type 1 isozyme (11beta-HSD-1) may function as an 11beta-reductase, regenerating active glucocorticoids from circulating inert 11-keto forms in specific tissues, notably the liver. To examine the importance of this enzyme isoform in vivo, mice were produced with targeted disruption of the 11beta-HSD-1 gene. These mice were unable to convert inert 11-dehydrocorticosterone to corticosterone in vivo. Despite compensatory adrenal hyperplasia and increased adrenal secretion of corticosterone, on starvation homozygous mutants had attenuated activation of the key hepatic gluconeogenic enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, presumably, because of relative intrahepatic glucocorticoid deficiency. The 11beta-HSD-1 -/- mice were found to resist hyperglycamia provoked by obesity or stress. Attenuation of hepatic 11beta-HSD-1 may provide a novel approach to the regulation of gluconeogenesis.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1997

Demonstration of a metabolically active glucose-6-phosphate pool in the lumen of liver microsomal vesicles.

Glucose-6-phosphate transport was investigated in rat or human liver microsomal vesicles using rapid filtration and light-scattering methods. Upon addition of glucose-6-phosphate, rat liver microsomes accumulated the radioactive tracer, reaching a steady-state level of uptake. In this phase, the majority of the accumulated tracer was glucose, but a significant intraluminal glucose-6-phosphate pool could also be observed. The extent of the intravesicular glucose pool was proportional with glucose-6-phosphatase activity. The relative size of the intravesicular glucose-6-phosphate pool (irrespective of the concentration of the extravesicular concentration of added glucose-6-phosphate) expressed as the apparent intravesicular space of the hexose phosphate was inversely dependent on glucose-6-phosphatase activity. The increase of hydrolysis by elevating the extravesicular glucose-6-phosphate concentration or temperature resulted in lower apparent intravesicular glucose-6-phosphate spaces and, thus, in a higher transmembrane gradient of glucose-6-phosphate concentrations. In contrast, inhibition of glucose-6-phosphate hydrolysis by vanadate, inactivation of glucose-6-phosphatase by acidic pH, or genetically determined low or absent glucose-6-phosphatase activity in human hepatic microsomes of patients suffering from glycogen storage disease type 1a led to relatively high intravesicular glucose-6-phosphate levels. Glucose-6-phosphate transport investigated by light-scattering technique resulted in similar traces in control and vanadate-treated rat microsomes as well as in microsomes from human patients with glycogen storage disease type 1a. It is concluded that liver microsomes take up glucose-6-phosphate, constituting a pool directly accessible to intraluminal glucose-6-phosphatase activity. In addition, normal glucose-6-phosphate uptake can take place in the absence of the glucose-6-phosphatase enzyme protein, confirming the existence of separate transport proteins.

Animals↗
PubMed1997

Hepatic glucose-6-phosphatase development in preterm and full-term guinea-pigs: comparison with rat and human development.

In term infants, hepatic glucose-6-phosphatase activity rises several-fold in the first few days after birth. In contrast, in many preterm infants, the postnatal rise in activity does not occur and the abnormally low levels can persist. In an attempt to determine if prematurity causes long-term changes in levels of glucose-6-phosphatase in liver of all mammals, we have studied the ontogeny of glucose-6-phosphatase in term guinea-pigs, and also in guinea-pigs delivered prematurely by Caesarean section. The activity of hepatic glucose-6-phosphatase in preterm guinea-pigs is about 5-fold lower than in term guinea-pigs at birth but the activity rises rapidly to very similar values to those found in term guinea-pigs. This indicates that prematurity alone does not result in abnormal development of hepatic glucose-6-phosphatase activity in guinea pigs. The changes in liver glucose-6-phosphatase activity in the postnatal period in term guinea-pigs were much smaller than those occurring in term postnatal rats or term infants.

Age Factors↗