Identification of a cAMP response element within the glucose- 6-phosphatase hydrolytic subunit gene promoter which is involved in the transcriptional regulation by cAMP and glucocorticoids in H4IIE hepatoma cells.
(1/767)
The expression of a luciferase reporter gene under the control of the human glucose 6-phosphatase gene promoter was stimulated by both dexamethasone and dibutyryl cAMP in H4IIE hepatoma cells. A cis-active element located between nucleotides -161 and -152 in the glucose 6-phosphatase gene promoter was identified and found to be necessary for both basal reporter-gene expression and induction of expression by both dibutyryl cAMP and dexamethasone. Nucleotides -161 to -152 were functionally replaced by the consensus sequence for a cAMP response element. An antibody against the cAMP response element-binding protein caused a supershift in gel-electrophoretic-mobility-shift assays using an oligonucleotide probe representing the glucose 6-phosphatase gene promoter from nucleotides -161 to -152. These results strongly indicate that in H4IIE cells the glucose 6-phosphatase gene-promoter sequence from -161 to -152 is a cAMP response element which is important for the regulation of transcription of the glucose 6-phosphatase gene by both cAMP and glucocorticoids. (+info)
Molecular cloning of a pancreatic islet-specific glucose-6-phosphatase catalytic subunit-related protein.
(2/767)
A pancreatic islet-specific glucose-6-phosphatase-related protein (IGRP) was cloned using a subtractive cDNA expression cloning procedure from mouse insulinoma tissue. Two alternatively spliced variants that differed by the presence or absence of a 118-bp exon (exon IV) were detected in normal balb/c mice, diabetic ob/ob mice, and insulinoma tissue. The longer, 1901-bp full-length cDNA encoded a 355-amino acid protein (molecular weight 40,684) structurally related (50% overall identity) to the liver glucose-6-phosphatase and exhibited similar predicted transmembrane topology, conservation of catalytically important residues, and the presence of an endoplasmic reticulum retention signal. The shorter transcript encoded two possible open reading frames (ORFs), neither of which possessed His174, a residue thought to be the phosphoryl acceptor (Pan CJ, Lei KJ, Annabi B, Hemrika W, Chou JY: Transmembrane topology of glucose-6-phosphatase. J Biol Chem 273:6144-6148, 1998). Northern blot and reverse transcription-polymerase chain reaction analysis showed that the mRNA was highly expressed in pancreatic islets and expressed more in beta-cell lines than in an alpha-cell line. It was notably absent in tissues and cell lines of non-islet neuroendocrine origin, and no other major tissue source of the mRNA was found. During development, it was expressed in parallel with insulin mRNA. The mRNA was efficiently translated and glycosylated in an in vitro translation/membrane translocation system and readily transcribed into COS 1, HIT, and CHO cells using cytomegalovirus or Rous sarcoma virus promoters. Whereas the liver glucose-6-phosphatase showed activity in these transfection systems, the IGRP failed to show glucose phosphotransferase or phosphatase activity with p-nitrophenol phosphate, inorganic pyrophosphate, or a range of sugar phosphates hydrolyzed by the liver enzyme. While the metabolic function of the enzyme is not resolved, its remarkable tissue-specific expression warrants further investigation, as does its transcriptional regulation in conditions where glucose responsiveness of the pancreatic islet is altered. (+info)
Structure and promoter activity of an islet-specific glucose-6-phosphatase catalytic subunit-related gene.
(3/767)
In liver and kidney, the terminal step in the gluconeogenic pathway is catalyzed by glucose-6-phosphatase (G-6-Pase). This enzyme is actually a multicomponent system, the catalytic subunit of which was recently cloned. Numerous reports have also described the presence of G-6-Pase activity in islets, although the role of G-6-Pase in this tissue is unclear. Arden and associates have described the cloning of a novel cDNA that encodes an islet-specific G-6-Pase catalytic subunit-related protein (IGRP) (Arden SD, Zahn T, Steegers S, Webb S, Bergman B, O'Brien RM, Hutton JC: Molecular cloning of a pancreatic islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP). Diabetes 48:531-542, 1999). We screened a mouse BAC library with this cDNA to isolate the IGRP gene, which spans approximately 8 kbp of genomic DNA. The exon/intron structure of the IGRP gene has been mapped and, as with the gene encoding the liver/kidney G-6-Pase catalytic subunit, it is composed of five exons. The sizes of these exons are 254 (I), 110 (II), 112 (III), 116 (IV), and 1284 (V) bp, similar to those of the G-6-Pase catalytic subunit gene. Two interspecific backcross DNA mapping panels were used to unambiguously localize the IGRP gene (map symbol G6pc-rs) to the proximal portion of mouse chromosome 2. The IGRP gene transcription start site was mapped by primer extension analysis, and the activity of the IGRP gene promoter was analyzed in both the islet-derived HIT cell line and the liver-derived HepG2 cell line. The IGRP and G-6-Pase catalytic subunit gene promoters show a reciprocal pattern of activity, with the IGRP promoter being approximately 150-fold more active than the G-6-Pase promoter in HIT cells. (+info)
Identification of protein components of the microsomal glucose 6-phosphate transporter by photoaffinity labelling.
(4/767)
The glucose-6-phosphatase system catalyses the terminal step of hepatic glucose production from both gluconeogenesis and glycogenolysis and is thus a key regulatory factor of blood glucose homoeostasis. To identify the glucose 6-phosphate transporter T1, we have performed photoaffinity labelling of human and rat liver microsomes by using the specific photoreactive glucose-6-phosphate translocase inhibitors S 0957 and S 1743. Membrane proteins of molecular mass 70, 55, 33 and 31 kDa were labelled in human microsomes by [3H]S 0957, whereas in rat liver microsomes bands at 95, 70, 57, 54, 50, 41, 33 and 31 kDa were detectable. The photoprobe [3H]S 1743 led to the predominant labelling of a 57 kDa and a 50 kDa protein in the rat. Stripping of microsomes with 0.3% CHAPS retains the specific binding of T1 inhibitors; photoaffinity labelling of such CHAPS-treated microsomes resulted in the labelling of membrane proteins of molecular mass 55, 33 and 31 kDa in human liver and 50, 33 and 31 kDa in rat liver. Photoaffinity labelling of human liver tissue samples from a healthy individual and from liver samples of patients with a diagnosed glycogen-storage disease type 1b (GSD type 1b; von Gierke's disease) revealed the absence of the 55 kDa protein from one of the patients with GSD type 1. These findings support the identity of the glucose 6-phosphate transporter T1, with endoplasmic reticulum protein of molecular mass 50 kDa in rat liver and 55 kDa in human liver. (+info)
Quantitative aspects of relationship between glucose 6-phosphate transport and hydrolysis for liver microsomal glucose-6-phosphatase system. Selective thermal inactivation of catalytic component in situ at acid pH.
(5/767)
Studies of the thermal stability of rat liver glucose-6-phosphatase (EC 3.1.3.9) were carried out to further elevate the proposal that the enzymic activity is the result of the coupling of a glucose-6-P-specific translocase and a nonspecific phosphohydrolase-phosphotransferase. Inactivation was observed when micorsomes were incubated at mild temperatures between pH 6.2 and 5.6. The rate of inactivation increased either with increasing hydrogen ion concentration or temperature. However, no inactivation was seen below 15 degrees in media as low as pH 5 or at neutral pH up to 37 degrees. The thermal stability of the enzyme may be controlled by the physical state of the membrane lipids and the degree of protonation of specific residues in the enzyme protein. Microsomes were exposed to inactivating conditions, and kinetic analyses were made of the glucose-6-P phosphohydrolase activities before and after supplementation to 0.4% sodium taurocholate. The results support the postulate and the kinetic characteristics of a given preparation of intact microsomes are determined by the relative capacities of the transport and catalytic components. Before detergent treatment, inactivation (i.e. a decrease in Vmax) was accompanied by a decrease in Km and a reduction in the fraction of latent activity, whereas only Vmax was depressed in disrupted preparations. The possibility that the inactivating treatments caused concurrent disruption of the microsomal membrane was ruled out. It is concluded that exposures to mild heat in acidic media selectively inactivate the catalytic component of the glucose-6-phosphatase system while preserving an intact permeability barrier and a functional glucose-6-P transport system. Analyses of kinetic data obtained in the present and earlier studies revealed several fundamental mathematical relationships among the kinetic constants describing the glucose-6-P phosphohydrolase activities of intact (i.e. the "system") and disrupted microsomes (i.e. the catalytic component). The quantitative relationships appear to provide a means to calculate a velocity constant (VT) and a half-saturation constant (KT) for glucose-6-P influx. The well documented, differential responses of the rat liver glucose-6-phosphatase system induced by starvation, experimental diabetes, or cortisol administration were analyzed in terms of these relationships. The possible influences of cisternal inorganic phosphate on the apparent kinetic constants of the intact system are discussed. (+info)
The effect of connexin32 null mutation on hepatocarcinogenesis in different mouse strains.
(6/767)
Connexin32 (Cx32) is the major gap junctional protein in mouse liver. We have shown recently that the formation of liver tumours in Cx32-deficient mice is strongly increased in comparison with control wild-type mice, demonstrating that the deficiency in gap junctional communication has an enhancing effect on hepatocarcinogenesis. We have now compared the effect of Cx32 deficiency on liver carcinogenesis in two strains of mice with differing susceptibility to hepatocarcinogenesis. Heterozygous Cx32(+/-) females were crossed with male Cx32 wild-type C57BL/6J (low susceptibility) or C3H/He (high susceptibility) mice. Since the Cx32 gene is located on the X-chromosome, the resulting F1 males segregated to the genotypes Cx32(Y/+) and Cx32(Y/-). Genotyping was performed by PCR-analysis using tail-tip DNA. Weanling male mice were i.p. injected with a single dose of N-nitrosodiethylamine and were killed 16, 21 or 26 weeks later. The number, volume fraction and size distribution of precancerous liver lesions characterized by a deficiency in the marker enzyme glucose-6-phosphatase were quantitated. The results demonstrate that Cx32 deficiency only slightly affects the number of enzyme-altered lesions, but strongly enhances their growth, both in the resistant and the susceptible mouse strain, suggesting that decreased intercellular communication results in tumour promoting activity irrespective of the genetic background of the mouse strain used. Since Cx32-deficient C3H/He hybrids were approximately 5-10 times more sensitive than C3H/He hybrids with an intact Cx32 gene, this mouse strain may prove very useful for toxicological screening purposes. (+info)
Comparison between in vitro lipid peroxidation in fresh sheep platelets and peroxidative processes during sheep platelet ageing under storage at 4 degrees C.
(7/767)
Incubation of sheep platelet crude membranes with xanthine oxidase (XO)/hypoxanthine/Fe(2+)-ADP revealed: (i) a fast peroxidative response - with a maximal linear rate of 14 nmol malondialdehyde (MDA) equivalents/mg protein, as evidenced by the thiobarbituric acid test - and a decrease in the polyunsaturated fatty acid (PUFA) content of the platelet crude membranes; (ii) a decrease in the lipid fluidity in the deep lipid core of the membranes but not at the membrane surface; (iii) a dramatic inhibitory effect on glucose 6-phosphatase (Glc-6-Pase) but not on acetylcholinesterase activity. Platelets were also aged by storage at 4 degrees C in their own plasma or in Seto additive solution. In these media, platelet aggregates were visible and the effects on platelet phospholipids, PUFA, lipid extract fluorescence, crude membrane fluidity and membrane-bound enzyme activities were assessed for comparison with those observed in in vitro lipid peroxidation. The sensitivity of membranes from stored platelets to lipid peroxidation was also assessed. Storage of platelets in plasma for 5 days was associated with different changes in their crude membranes such as decreases in arachidonic acid contents, the decrease not being avoided by the presence of phospholipase A(2) inhibitors, increases in MDA equivalents, conjugated dienes and lipid extract fluorescence, decreases in the amounts of MDA equivalents formed by platelet crude membranes treated with the oxidizing agents, changes in membrane fluidity and inhibition of Glc-6-Pase. All these alterations were less pronounced or even abolished after platelet storage in Seto. These findings suggest that platelet lipid peroxidation due to XO/hypoxanthine/Fe(2+)-ADP and platelet membrane alterations observed after platelet ageing under storage at 4 degrees C share common features. Also, as regards the prevention of peroxidative processes, Seto solution permits better storage of sheep platelets than plasma. (+info)
Dehydroepiandrosterone suppresses the elevated hepatic glucose-6-phosphatase and fructose-1,6-bisphosphatase activities in C57BL/Ksj-db/db mice: comparison with troglitazone.
(8/767)
The effect of dehydroepiandrosterone (DHEA) on the hepatic and muscle glucose metabolizing enzymes and on blood glucose were investigated in insulin-resistant diabetic C57BL/KsJ-db/db mice and their heterozygote littermates (db/+m). The results were compared with those after troglitazone administration under the same conditions. Despite hyperinsulinemia, hepatic glucose-6-phosphatase (G6Pase) and fructose-1,6-bisphosphatase (FBPase) activities are higher in db/db than in db/+m mice. Dietary administration of DHEA and that of troglitazone for 15 days to respective groups of five mice each significantly decreased blood glucose in db/db mice and hepatic G6Pase and FBPase activities in both db/db and db/+m mice. Hepatic G6Pase and FBPase activities showed a linear relationship with blood glucose in all groups of mice, suggesting that the activities of G6Pase and FBPase are closely related to blood glucose levels. Because androstenedione, a DHEA metabolite, barely affected either of these enzyme activities or blood glucose in db/db mice, the actions of DHEA, which are similar to those of troglitazone, are presumed to be caused by DHEA itself. DHEA is considered to be a modulating agent for the activities of hepatic gluconeogenic enzymes in db/db mice. (+info)