Ann Burchell

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.

Identification of the Ca2+‐dependent modulator protein as the fourth subunit of rabbit skeletal muscle phosphorylase kinase

Kakuichi et al. [l] and Cheung [2,3] were the first to demonstrate the presence of a factor in brain homogenates, which in the presence of Ca”‘, stimulated the activity of one of the cyclic nucleotide phosphodiesterases of this tissue. This factor was subsequently shown to be a small heat stable calcium binding protein, which was present in high concentrations in a wide variety of animal tissues [4-61. Following its purification to apparent homogeneity from bovine brain [7] and bovine heart [8] , it was noted that its physico-chemical properties were very similar to the calcium-binding subunit of rabbit skeletal muscle troponin, troponln-C, the protein which confers calcium sensitivity to actomyosin ATPase [9,10] . This idea was substantiated by the determination of the amino acid sequence of the ‘calcium-dependent modulator’ from bovine brain [ 1 l] and rat testis [ 121, which showed extensive homology with troponin-C, and by the finding that the ‘modulator’ could substitute for troponin-C in restoring calcium sensitivity to actomyosin ATPase in reconstituted systems [ 131. Troponin-C can also substitute for the ‘modulator’ in the activation of cyclic nucleotide

A new microtechnique for the analysis of the human hepatic microsomal glucose-6-phosphatase system

A microtechnique has been developed which enables a complete kinetic analysis of the human hepatic microsomal glucose-6-phosphatase system to be carried out in microsomes isolated from very small liver samples. Complete or partial deficiencies of any of the proteins of the glucose-6-phosphatase system resulting in Type 1a, 1b, 1c or 1d glycogen storage disease can be therefore be diagnosed using hepatic needle biopsy samples, whereas previous methods of diagnosis needed large wedge biopsy samples requiring laparotomy.

Cloning and sequencing of the 5' region of the human glucose-6-phosphatase gene: transcriptional regulation by cAMP, insulin and glucocorticoids in H4IIE hepatoma cells.

We have cloned and sequenced the first 1.2 kb of the 5′ region of the human glucose‐6‐phosphatase gene. Transfection of H4IIE hepatoma cells with the 1.2 kb fragment fused to a luciferase reporter gene demonstrated both basal and hormone responsive luciferase activity. Dexamethasone increased and insulin decreased luciferase activity. Insulin and dibutyryl cyclic AMP both significantly decreased activity in the presence of dexamethasone.

The molecular basis of the hepatic microsomal glucose-6-phosphatase system

Role, regulation et deficit genetique de chacune des 5 proteines constituant le systeme enzymatique de la glucose-6-phosphatase: sous-unite catalytique, proteine de liaison au calcium (proteine SP), et 3 proteines de transport (T 1 , T 2 , T 3 )

A Role for Astrocytes in Glucose Delivery to Neurons

The present paper examines the possible role of astrocytes in the delivery of glycogen-derived glucose for neuronal metabolism. Such a process would require astrocytic expression of glucose-6-phosphatase. The degree and significance of brain expression of glucose-6-phosphatase (EC 3.1.3.9) has been a subject of controversy. Published immunohistochemical data are consistent with expression of glucose-6-phosphatase by astrocytes, both in vivo and in vitro. In this paper additional confirmation of the expression of glucose-6-phosphatase mRNA in rat brain is presented. Although cultured astrocytes demonstrate glucose-6-phosphatase activity in vitro under assay conditions, there is very limited in vitro evidence that this activity confers a glucose-export capacity on astrocytes. Under most conditions in vitro, lactate export predominates, however this may relate to aspects of the in vitro phenotype. Data relating to astrocytic glucose and lactate export are considered in the context of hypotheses of trafficking by astrocytes of substrates for neuronal metabolism, hypotheses that imply and require compartmentation of these substances, in contrast with current formulations of glucose transport into and within brain that imply no glucose compartmentation. Microdialysis studies of the properties of the brain extracellular fluid (ECF) glucose pool in the freely moving rat were performed seeking evidence of glucose compartmentation. Results of these studies do imply compartmentalisation of brain glucose, and are consistent with a model envisaging the majority of glucose reaching the neuron via the astrocytic intracellular space and the ECF. In addition, such studies provide evidence that rises in ECF glucose concentration are not the direct result of local recruitment of cerebral blood flow, but suggest the influence of intermediate, astrocyte-based mechanisms. Astrocytic glucose-6-phosphatase may permit astrocytes to modulate the trans-astrocytic flux of glucose to adjacent neurons in response to signals reflecting increased neuronal demand.

Identification and characterisation of a new human glucose-6-phosphatase isoform.

The liver endoplasmic reticulum glucose‐6‐phosphatase catalytic subunit (G6PC1) catalyses glucose 6‐phosphate hydrolysis during gluconeogenesis and glycogenolysis. The highest glucose‐6‐phosphatase activities are found in the liver and the kidney; there have been many reports of glucose 6‐phosphate hydrolysis in other tissues. We cloned a new G6Pase isoform (G6PC3) from human brain encoded by a six‐exon gene (chromosome 17q21). G6PC3 protein was able to hydrolyse glucose 6‐phosphate in transfected Chinese hamster ovary cells. The optimal pH for glucose 6‐phosphate hydrolysis was lower and the K m higher relative to G6PC1. G6PC3 preferentially hydrolyzed other substrates including pNPP and 2‐deoxy‐glucose‐6‐phosphate compared to the liver enzyme.

DIAGNOSIS OF TYPE 1a AND TYPE 1c GLYCOGEN STORAGE DISEASES IN ADULTS

The hepatic glucose-6-phosphatase system was studied with a novel microanalytical technique in adult patients undergoing liver biopsy. 4 patients were diagnosed as having type 1 glycogen storage disease (GSD). 3 of these patients, who had hypoglycaemic symptoms, had variations of type 1a GSD, which is caused by a defect in the hepatic microsomal glucose-6-phosphatase enzyme. The fourth, with hepatomegaly and no hypoglycaemic symptoms, had a normal glucose-6-phosphatase enzyme but a defect in the hepatic microsomal phosphate/pyrophosphate translocase T2; this is the first report of an adult with type 1c GSD. Adult type 1 GSD should be considered in patients with unresolved hypoglycaemic symptoms and/or unresolved hepatomegaly.

Glucose-6-phosphatase proteins of the endoplasmic reticulum (Review)

Hepatic glucose-6-phosphatase (G-6-Pase) catalyses the terminal step of hepatic glucose production and it plays a key role in the maintenance of blood glucose homeostasis. Hepatic G-6-Pase is an integral resident endoplasmic reticulum (ER) protein and it is part of a multicomponent system. Its active site is situated inside the lumen of the ER and transport proteins are needed to allow its substrates, glucose-6-phosphate (G-6-P) (and pyrophosphate), and its products, phosphate and glucose to cross the ER membrane. In addition, a calcium-binding protein is also associated with the G-6-Pase enzyme. Recent immunological studies have shown that G-6-Pase (which has conventionally been thought to be present only in the gluconeogenic organs) is present in minor cell types in a variety of human tissues and that its distribution changes dramatically during human development. In all the tissues, enzymatic analysis, direct transport assays and/or immunological detection of the ER glucose and phosphate transport proteins have been used to demonstrate the presence and activity of the whole G-6-Pase system. The G-6-Pase protein is very hydrophobic and has proved difficult to purify to homogeneity. Four proteins of the system have now been isolated and polyclonal antibodies have been raised against them; two have also been cloned. The available sequences, together with topological studies, have given some information about both the topology of the proteins in the ER and the probable mechanisms by which the proteins are retained in the ER.

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.