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A Behrooz

Publications and source records attributed to A Behrooz.

10 recordsLinked to original sources
PubMed2000

Regulation of glut1 mRNA by hypoxia-inducible factor-1. Interaction between H-ras and hypoxia.

Oncogenic transformation and hypoxia both induce glut1 mRNA. We studied the interaction between the ras oncogene and hypoxia in up-regulating glut1 mRNA levels using Rat1 fibroblasts transformed with H-ras (Rat1-ras). Transformation with H-ras led to a substantial increase in glut1 mRNA levels under normoxic conditions and additively increased glut1 mRNA levels in concert with hypoxia. Using a luciferase reporter construct containing 6 kilobase pairs of the glut1 promoter, we showed that this effect was mediated at the transcriptional level. Promoter activity was much higher in Rat1-ras cells than in Rat1 cells and could be down-regulated by cotransfection with a dominant negative Ras construct (RasN17). A 480-base pair (bp) cobalt/hypoxia-responsive fragment of the promoter containing a HIF-1 binding site showed significantly higher activity in Rat1-ras cells than in Rat1 cells, suggesting that Ras might mediate its effect through HIF-1 even under normoxic conditions. Consistent with this, Rat1-ras cells displayed higher levels of HIF1-alpha protein under normoxic conditions. In addition, a promoter construct containing a 4-bp mutation in the HIF1 binding site showed lower activity in Rat1-ras cells than a construct with an intact HIF1 binding site. The activity of the latter construct but not the former could be down-regulated by RasN17, supporting the importance of the HIF1 binding site in regulation by Ras. The phosphatidylinositol 3-kinase inhibitor LY29004 down-regulated glut1 promoter activity and mRNA levels under normoxia and also decreased HIF1alpha protein levels in these cells. Collectively these results indicate that H-Ras up-regulates the glut1 promoter, at least in part, by increasing HIF-1alpha protein levels leading to transactivation of promoter through the HIF-1 binding site.

Animals↗
PubMed2000

An NF-Y binding site is important for basal, but not gonadotropin-releasing hormone-stimulated, expression of the luteinizing hormone beta subunit gene.

Regulated synthesis of luteinizing hormone (LH) requires coordinated transcriptional control of the alpha and LHbeta subunits in pituitary gonadotropes. Several cis-acting elements and trans-acting factors have been defined for control of the LHbeta promoter through heterologous cell culture models. In this report, we describe the identification of bipartite NF-Y (CBF/CP1) binding sites within the proximal bovine LHbeta promoter. When multimerized, one of these sites activates the heterologous, minimal HSV thymidine kinase promoter in the gonadotrope-derived cell line alphaT3-1. The functional role of the promoter-distal site in regulating the full-length bovine LHbeta promoter was assessed in vivo using transgenic mice harboring a mutant promoter linked to the chloramphenicol acetyltransferase reporter gene. While this element is important for conferring high level activity of the LHbeta promoter in pituitary, it does not appear to be essential for mediating gonadotropin-releasing hormone (GnRH) regulation. This is the first characterization of a cis-acting element within this GnRH-dependent promoter that is restricted to regulating basal expression and not GnRH-induced activity.

Animals↗
PubMed1999

LY-83583 stimulates glucose transporter-1-mediated glucose transport independent of changes in cGMP levels.

Exposure of Clone 9 cells, a nontransformed rat liver cell line expressing only the Glutl glucose transporter isoform, to the guanylyl cyclase inhibitor LY-83583 was found to stimulate the rate of glucose transport (approximately 7- to 8-fold in 1 h). A similar response to LY-83583 was found in NIH 3T3 fibroblasts, 3T3-L1 pre-adipocytes, and C2C12 myoblasts. Neither the rate of glucose transport in cells under control conditions nor the effect of LY-83583 on glucose transport was altered by 10, 50, or 100 microM 8-bromo-cGMP or by addition of cGMP phosphodiesterase inhibitors, zaprinast, or dipyridamole suggesting that glucose transport and the response to LY-83583 is independent of cGMP levels. In addition, the effect of LY-83583 on glucose transport was not mediated by inhibition of oxidative phosphorylation, since exposure to the agent resulted in no increase in lactate production. Incubation of Clone 9 cells in the presence of the phospholipase C inhibitor U73122, however, attenuated the glucose transport response to LY-83583. Moreover, exposure to LY-83583 resulted in a rise in cell diacylglycerol content, and preincubation with U73122 significantly diminished this rise as well as the glucose transport response to LY-83583. The stimulatory effect of LY-83583 on glucose transport was significantly blocked by thapsigargin. Down-regulation of protein kinase C activity, resulting from 24 h pre-incubation in the presence of 160 nM phorbol-12-myristate 13-acetate, did not attenuate the glucose transport response to LY-83583. It is concluded that the stimulation of glucose transport in response to LY-83583 is independent of changes in cGMP levels, is not mediated by inhibition of oxidative phosphorylation, and is mediated, at least in part, through stimulation of the phospholipase C pathway.

3-O-Methylglucose↗
PubMed1999

Regulation of glucose transport by hypoxia.

Transport of glucose into most mammalian cells and tissues is rate-controlling for its metabolism. Glucose transport is acutely stimulated by hypoxic conditions, and the response is mediated by enhanced function of the facilitative glucose transporters (Glut), Glut1, Glut3, and Glut4. The expression and activity of the Glut-mediated transport is coupled to the energetic status of the cell, such that the inhibition of oxidative phosphorylation resulting from exposure to hypoxia leads to a stimulation of glucose transport. The premise that the glucose transport response to hypoxia is secondary to inhibition of mitochondrial function is supported by the finding that exposure of a variety of cells and tissues to agents such as azide or cyanide, in the presence of oxygen, also leads to stimulation of glucose transport. The mechanisms underlying the acute stimulation of transport include translocation of Gluts to the plasma membrane (Glut1 and Glut4) and activation of transporters pre-exiting in the plasma membrane (Glut1). A more prolonged exposure to hypoxia results in enhanced transcription of the Glut1 glucose transporter gene, with little or no effect on transcription of other Glut genes. The transcriptional effect of hypoxia is mediated by dual mechanisms operating in parallel, namely, (1) enhancement of Glut1 gene transcription in response to a reduction in oxygen concentration per se, acting through the hypoxia-signaling pathway, and (2) stimulation of Glut1 transcription secondary to the associated inhibition of oxidative phosphorylation during hypoxia. Among the various hypoxia-responsive genes, Glut1 is the first gene whose rate of transcription has been shown to be dually regulated by hypoxia. In addition, inhibition of oxidative phosphorylation per se, and not the reduction in oxygen tension itself, results in a stabilization of Glut1 mRNA. The increase in cell Glut1 mRNA content, resulting from its enhanced transcription and decreased degradation, leads to increased cell and plasma membrane Glut1 content, which is manifested by a further stimulation of glucose transport during the adaptive response to prolonged exposure to hypoxia.

Animals↗
PubMed1998

Induction of GLUT1 mRNA in response to azide and inhibition of protein synthesis.

Incubation of Clone 9 cells, a nontransformed rat liver cell line, in the presence of 5 mM azide results in an induction of GLUT1 mRNA which becomes detectable after approximately 3 h of continuous exposure to the agent. In examining the role of on-going protein synthesis in this response, we found that: (i) the induction of GLUT1 mRNA by azide was not inhibited by anisomycin, (ii) exposure to anisomycin alone also resulted in increased GLUT1 mRNA content, and (iii) the increments in GLUT1 mRNA content in the presence of both azide and anisomycin were additive. Following exposure to 30 microM anisomycin, the increase in GLUT1 mRNA content became evident at 1 h, reached a maximum level of approximately 7-fold at 3 h, then slowly decreased but remained elevated at approximately 2-fold control levels at 12 h. Transcription of the GLUT1 gene, estimated by nuclear run-on assay, was stimulated 1.4 +/- 0.1 and 1.6 +/- 0.2-fold in cells exposed to anisomycin for 1 and 2 h, respectively (p < 0.05 for both). Upon inhibition of RNA synthesis by actinomycin D, GLUT1 mRNA content decreased with a half-life of 1.9 +/- 0.4 h in control cells, while in contrast, GLUT1 mRNA half-life was 4.6 +/- 0.8 h in cells exposed to anisomycin. The induction of GLUT1 mRNA by anisomycin was half-maximal at approximately 3 microM, whereas inhibition of leucine incorporation and stimulation of Stress Activated Protein Kinase (SAPK), measured as c-Jun N-terminal kinase activity, were half-maximal at approximately 0.3 and approximately 0.05 microM anisomycin, respectively. GLUT1 mRNA content was also increased by the protein synthesis inhibitor emetine, and the effect was associated with no stimulation of SAPK activity. Finally, SAPK activity was minimally stimulated in cells exposed to azide. It is concluded that: (1) on-going protein synthesis is not necessary for the induction of GLUT1 mRNA content in response to azide, (2) the induction of GLUT1 mRNA by anisomycin is related to its activity to inhibit protein synthesis, and (3) under basal conditions, a rapidly turning-over putative protein exerts a negative regulatory effect on GLUT1 mRNA expression.

Animals↗
PubMed1997

Glycemia-lowering effect of cobalt chloride in the diabetic rat: increased GLUT1 mRNA expression.

We have recently shown that expression of the GLUT1 glucose transporter isoform is augmented in cells exposed to cobalt chloride [Co(II)], an agent that stimulates the expression of hypoxia-responsive genes (Behrooz, A., Ismail-Beigi, F., 1997. J. Biol. Chem. 272, 5555-5562.). Here, we examine the effect of Co(II) on glycemia and tissue GLUT1 mRNA content of normal and diabetic rats. The addition of 2 mM Co(II) in the drinking water reduced the glycemia of streptozotocin-induced diabetic rats by day 3 from 32.3 +/- 2.1 to 21.0 +/- 1.9 mM (non-fasting). Co(II) resulted in no change in serum insulin levels of normal or diabetic rats. Treatment with 4 mM Co(II) was more effective than 2 mM Co(II) in reducing the glycemia of diabetic rats, while 6 mM Co(II) was associated with severe toxicity. GLUT1 mRNA content increased significantly in ventricular myocardium, renal cortex, skeletal muscle, cerebrum and liver of normal and diabetic rats treated with 2 mM cobalt chloride (ranging from 1.3- to 2.9-fold in the different tissues). It is concluded that: (1) treatment with Co(II) decreases the glycemia of diabetic rats, and (2) the glycemia-lowering effect of Co(II) is associated with, and may be mediated by, enhanced expression of GLUT1 mRNA.

Animals↗
PubMed1997

Dual control of glut1 glucose transporter gene expression by hypoxia and by inhibition of oxidative phosphorylation.

glut1 gene expression and glucose transport are stimulated in a variety of cells and tissues in response to hypoxia. glut1 is also up-regulated by inhibitors of oxidative phosphorylation (such as azide) in the presence of oxygen. Here, we test the hypothesis that hypoxia stimulates glut1 gene expression independent of its inhibitory effect on oxidative phosphorylation. We examined the effect of cobalt chloride, a known stimulator of genes responsive to reduced oxygen concentration per se, on GLUT1 expression under normoxic conditions and compared the results with the response to azide. Exposure of a rat liver cell line (Clone 9) to 250 microM cobalt chloride increases GLUT1 mRNA content, which becomes evident at 2 h, reaches a maximal value of approximately 12-fold at 8 h, and remains elevated at approximately 8-fold at 24 h. GLUT1 mRNA was the only GLUT isoform expressed in control cells and in cells exposed to cobalt chloride or azide. The induction of GLUT1 mRNA by cobalt chloride is associated with a approximately 10-fold stimulation of cytochalasin B-inhibitable 3-O-methyl-D-glucose transport at 24 h. In contrast to the rapid decrease in cell ATP levels and the stimulation of glucose transport in response to azide, cell ATP content and glucose transport remained unaltered during the initial 1-h period of exposure to cobalt chloride. The effect of cobalt chloride on GLUT1 mRNA content is mimicked by Ni(II) or Mn(II) but not by Fe(II). Employing actinomycin D, we found no increase in the approximately 1.5-h half-life of GLUT1 mRNA in cobalt chloride-treated cells, suggesting that the effect of cobalt chloride on GLUT1 mRNA content is largely mediated at the transcriptional level; in contrast, GLUT1 mRNA half-life increased to >8 h in azide-treated cells. In transient transfections we found that approximately 6 kilobase pairs (kbp) of 5'-flanking region of the rat glut1 promoter confers both cobalt chloride- and azide-inducibility to a reporter gene. Deletion of approximately 2, 500 base pairs (bp) from the 5' end of the approximately 6-kbp DNA fragment results in a reduction of the response to cobalt chloride and a complete loss of the response to azide. A 666-bp DNA segment located approximately 6.0 kbp upstream of the transcription start site was found to be necessary for the increase in reporter gene expression in response to azide, whereas a 480-bp segment located at approximately -3.5 kbp mediated the response to cobalt chloride. The 480-bp segment is highly homologous to the previously reported mouse glut1 enhancer and contains several potential regulatory elements, including a hypoxia-inducible element; an additional hypoxia-inducible element is present in the 666-bp segment. Our results suggest that glut1 gene expression is regulated in a dual fashion by hypoxia per se and in response to inhibition of oxidative phosphorylation.

3T3 Cells↗
PubMed1995

Stimulation of GLUT-1 glucose transporter expression in response to exposure to calcium ionophore A-23187.

We tested the hypothesis that an increase in cytosolic calcium concentration stimulates glucose transporter isoform (GLUT-1) gene expression. Exposure of a rat liver cell line (Clone 9) to 3 microM A-23187 for 12 h resulted in 3-, 5-, and 10-fold increases in cytochalasin B-inhibitable 3-O-methyl-D-glucose transport, GLUT-1 protein, and GLUT-1 mRNA content, respectively. The induction of GLUT-1 mRNA in response to A-23187 is not preceded by a significant decrease in cell ATP content. This induction is prevented by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in conjunction with ethylene glycol-bis(beta-aminoethyl ether)-N,N, N',N'-tetraacetic acid. To investigate the mechanism of GLUT-1 mRNA induction, we found that exposure to A-23187 stabilized GLUT-1 mRNA: with the employment of actinomycin D, GLUT-1 mRNA had a half-life of 1.5 and 5.5 h in control and A-23187-treated cells, respectively. In nuclear run-on assays, the rate of GLUT-1 gene transcription was stimulated 1.5- to 1.7-fold in nuclei isolated from cells exposed to A-23187 for either 30 min or 2 h. These results demonstrate that exposure to A-23187 stimulates GLUT-1 gene expression and that the increase in GLUT-1 mRNA content is mediated in part by enhanced GLUT-1 gene transcription as well as decreased GLUT-1 mRNA degradation. The increase in GLUT-1 mRNA content, in turn, is associated with increased cell GLUT-1 content and enhanced glucose transport.

Animals↗