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F Maher

Publications and source records attributed to F Maher.

At least 37 records · Page 2Linked to original sources
PubMed1994

The GLUT3 glucose transporter is the predominant isoform in primary cultured neurons: assessment by biosynthetic and photoaffinity labelling.

Cerebellar granule neurons in primary culture express increasing levels of two glucose transporter isoforms, GLUT1 and GLUT3, as they differentiate in vitro. We have determined the relative abundance of GLUT1 and GLUT3 in these neurons by three different labelling methods. (1) Photoaffinity cell surface labelling of neurons with an impermeant bis-mannose photolabel revealed 6-10-fold more GLUT3 than GLUT1 and dissociation constants (Kd) for the photolabel of 55-68 microM (GLUT3) and 146-169 microM (GLUT1). Binding to both transporters was inhibited by cytochalasin B. (2) Photoaffinity labelling of neuronal membranes with a permeant forskolin derivative showed 5.5-8-fold more GLUT3 than GLUT1, whereas in rat brain membranes containing both neuronal and glial membranes, GLUT3 and GLUT1 were detected in similar proportions. (3) Biosynthetic labelling of neurons with [35S]methionine and [35S]cysteine showed GLUT3 to be 6-10-fold more abundant than GLUT1. Thus GLUT3 is quantitatively the predominant glucose-transport isoform in cultured cerebellar granule neurons.

Affinity Labels↗
PubMed1994

Modulation of expression of glucose transporters GLUT3 and GLUT1 by potassium and N-methyl-D-aspartate in cultured cerebellar granule neurons.

Depolarization is known to stimulate neuronal oxidative metabolism. As glucose is the primary fuel for oxidative metabolism in the brain, the entry of glucose into neural cells is a potential control point for any regulatory events in brain metabolism. Therefore, the effects of depolarizing stimuli, high K+ and N-methyl-D-aspartate (NMDA), were examined on the functional expression of glucose transporter isoforms GLUT1 and GLUT3 in primary cultured cerebellar granule neurons. Higher levels of glucose transport activity were observed in neurons cultured in 25 mM KCl (K25) compared to those in 5 and 15 mM KCl (K5 and K15). The elevated glucose transport activity correlated with increased levels of GLUT3 protein and, to a lesser extent, GLUT1. Both GLUT3 and GLUT1 were regulated at the level of mRNA expression. Addition of NMDA to K5 and K15 cultures increased both glucose uptake and GLUT3 protein levels, with smaller changes in GLUT1. NMDA effects were not additive with K25 effects. All these changes were observed only with chronic exposure of neurons to high K+ or NMDA; no acute effects on glucose uptake or transporter expression were found. Thus, chronic depolarization of primary cerebellar granule neurons acts as a stimulus for the expression of the neuronal GLUT3 glucose transporter isoform.

Animals↗
PubMed1994

Glucose transporter proteins in brain.

Glucose is the principal energy source for the mammalian brain. The presence of glucose transport proteins is essential to supply glucose to the neurons and glia within the brain. At least three glucose transporter isoforms have now been identified, and are thought to play a significant role, in the brain. This review describes our current understanding of cell-specific glucose transporter expression in brain, which includes GLUT1 (55-kDa form) present at a high concentration at the blood-brain barrier as well as in parenchymal cells (45-kDa form), most likely in astrocytes, GLUT3 expressed in neurons, and GLUT5 in microglia. We discuss some potential implications of this glucose transporter heterogeneity for cerebral metabolic activity.

Animals↗
PubMed1994

Altered expression of GLUT-1 and GLUT-3 glucose transporters in neurohypophysis of water-deprived or diabetic rats.

Progressive dehydration due to water deprivation and streptozotocin diabetes both produce increased activity of the hypothalamoneurohypophysial system and enhanced vasopressin secretion. To determine whether enhanced metabolic activity affects glucose transporter protein expression, this study examined the effect of these conditions on 45-kDa GLUT-1 and the neuronal glucose transporter, GLUT-3, which mediate glucose transport in the rat neurohypophysis. Progressive water deprivation increased hematocrit, plasma electrolytes Na+ and Cl-, and vasopressin over 3 days, relative to the severity of dehydration. Plasma vasopressin increased threefold by 24 h, reaching 4.5-fold by 72 h. These changes were reflected in a 56 and 75% decrease in neurohypophysial vasopressin content by 48 and 72 h, respectively. Significant changes in glucose transporters were also observed at 48 and 72 h, with GLUT-1 increasing by 18 and 44% and GLUT-3 increasing by 42 and 55%, respectively. Streptozotocin-induced diabetes produced increases in hematocrit, plasma Cl-, and vasopressin, although the magnitude of these changes was less than with dehydration. There was a twofold increase in plasma vasopressin by 3 days, commensurate with the onset of overt diabetes, and a threefold increase by 2 wk. These changes were reflected in a 30 and 40% decline in neural lobe vasopressin content, respectively. Despite the difference in the magnitude of hormone response, GLUT-3 increased by the same amount (53%) as in dehydration. GLUT-1, however, was decreased 16% by 3 days and 25% by 1 and 2 wk of diabetes. Although the opposite effects on GLUT-1 may relate to differences in circulating insulin or glucose, this study is the first demonstration of increased expression of GLUT-3 in response to a common hypothalamic signal in these two conditions.

Animals↗
PubMed1993

Interaction of 7-bromoacetyl-7-desacetylforskolin, and alkylating derivative of forskolin, with bovine brain adenylyl cyclase and human erythrocyte glucose transporter.

7-Bromoacetyl-7-desacetylforskolin (BrAcFsk), an alkylating derivative of forskolin, activated adenylyl cyclase and irreversibly blocked high affinity forskolin binding sites in human platelet membranes and rat brain membranes (Laurenza et al., 1990). Photoincorporation of an iodinated arylazido derivative of forskolin, 125I-6-AIPP-Fsk, into adenylyl cyclase in bovine brain membranes was irreversibly inhibited by BrAcFsk but not by 1,9-dideoxy-BrAcFsk, suggesting that BrAcFsk was reacting specifically with a nucleophilic group(s) at the forskolin binding site of adenylyl cyclase. Immunoblotting with antiforskolin antiserum demonstrated that partially purified bovine brain adenylyl cyclase had incorporated BrAcFsk. The interaction of BrAcFsk with the glucose transporter in human erythrocyte membranes was examined in a similar manner. Photoincorporation of 125I-7-AIPP-Fsk, an iodinated arylazido derivative of forskolin which is specific for the glucose transporter, into the glucose transporter was not irreversibly inhibited by BrAcFsk, suggesting that, in contrast to adenylyl cyclase, there is no reactive nucleophilic group at the forskolin binding site on the human erythrocyte glucose transporter. The immunoblotting procedure with antiforskolin antiserum confirmed that BrAcFsk was not covalently attached to human erythrocyte glucose transporter.

Adenylyl Cyclases↗
PubMed1993

Glucose transporter isoforms in brain: absence of GLUT3 from the blood-brain barrier.

Two glucose transporter (GLUT) isoforms have been identified in brain. The GLUT1 isoform is abundant in cerebral microvessels and may be present in glia and neurons, whereas GLUT3 is probably the major neuronal glucose transporter. This study investigates whether GLUT3 is also present in microvessels from rat, human, and canine brain, by means of antisera directed against the divergent C-terminal sequences of mouse and human GLUT3. GLUT1 was detected in whole brain as two molecular mass forms: 55 kDa in microvessels and 45 kDa in cortical neuronal/glial membranes. With the aid of the appropriate antisera to the species-specific sequences, GLUT3 was detected in rat and human cortical membranes but not in isolated rat or human microvessels. These antisera failed to detect GLUT3 in either canine cortical membranes or canine microvessels, implying additional species specificity in the C-terminal sequence.

Amino Acid Sequence↗
PubMed1992

Detection of the GLUT3 facilitative glucose transporter in rat L6 muscle cells: regulation by cellular differentiation, insulin and insulin-like growth factor-I.

The GLUT3 facilitative glucose transporter protein was found to be expressed in rat L6 muscle cells. It was detected at both the myoblast and myotube stage. GLUT3 protein content per mg of total membrane protein increased significantly during L6 cell differentiation. Subcellular fractionation demonstrated that the GLUT3 protein was predominantly localized in plasma membrane-enriched fractions of either myoblasts or myotubes. Short-term exposure of L6 myotubes to IGF-I or insulin caused a redistribution of GLUT3 protein from an intracellular membrane fraction to the plasma membrane, without affecting total membrane GLUT3 protein content. Long-term exposure of L6 myotubes to IGF-I produced an increase of GLUT3 protein in total membranes and all subcellular membrane fractions, especially the plasma membrane. We propose that the GLUT3 glucose transporter may play an important role in glucose metabolism in developing muscle.

Animals↗
PubMed1992

Expression of mouse-GLUT3 and human-GLUT3 glucose transporter proteins in brain.

Polyclonal anti-peptide antisera were raised to the C-terminal sequence of mouse- and human-GLUT3 glucose transporter isoforms. GLUT3 protein and mRNA expression were investigated by Western blot and Northern blot assays, in a range of tissues and cell lines. Mouse-GLUT3 protein was detected only in rat brain, where it was present in most regions except adenohypophysis and pineal gland. Mouse-GLUT3 was also detected in primary cultured rat cerebellar neurons and the neuronal cell lines PC12 and NG108-15, but not in cultured astroglia. Human-GLUT3 protein was detected in CHO cells transfected with the human-GLUT3 cDNA and in human brain, but not in human placenta or fat. The expression of GLUT3 in rat brain and neurons indicates it is a major neuronal glucose transporter.

Amino Acid Sequence↗
PubMed1991

Stimulation of glucose transporter (GLUT1) mRNA and protein expression by inhibitors of glycosylation.

Glucose deprivation increases the steady-state levels of mRNA for the rat brain/HepG2-type glucose transporter (GLUT1) in L6 myocytes. Glucose deprivation also inhibits N-linked glycosylation. We therefore investigated a possible relationship between inhibition of glycosylation and GLUT1 expression in cultured L6 myocytes by determining the effects on GLUT1 expression of known inhibitors of glycosylation, namely tunicamycin, 2-deoxyglucose and glucosamine. All conditions prevented incorporation of [3H]mannose into TCA-precipitable myocyte protein and resulted in a 2- to 5-fold increase in the level of GLUT1 mRNA detected on Northern blots. Glucose deprivation and tunicamycin treatment caused an approx. 2-fold increase in GLUT1 mRNA half-life. GLUT1 protein, detected on immunoblots, accumulated 10- to 20-fold in response to all glycosylation inhibitors, with apparent molecular masses of 40 kDa after glucose deprivation, 42 kDa after 2-deoxyglucose and 38 kDa after glucosamine or tunicamycin treatments, compared to 45-50 kDa in glucose-fed cells. However, glucose deprivation was the only condition in which the rate of 2-deoxy-[3H]glucose uptake increased (3- to 5-fold). These results demonstrate a direct correlation between inhibition of glycosylation and the induction of GLUT1 mRNA and protein expression and suggest that the stability of GLUT1 mRNA is controlled by a signal associated with glycosylation.

Animals↗
PubMed1990

Stabilization of glucose transporter mRNA by insulin/IGF-1 and glucose deprivation.

Chronic exposure of L6 myocytes to insulin/IGF-1 or glucose deprivation results in an increase in the level of brain-type glucose transporter (GLUT1) mRNA. We have investigated the effects of insulin and glucose deprivation on GLUT1 mRNA stability. The half-life of GLUT1 mRNA in control cells was 2-2.5 h. Insulin increased GLUT1 mRNA levels by 5- to 10-fold, and GLUT1 mRNA half-life and transcription by 2-fold. Glucose deprivation increased GLUT1 mRNA level by 2- to 4-fold and half-life by 2-fold. The effects of insulin and glucose deprivation on GLUT1 mRNA stability were additive. Cycloheximide partially blocked the induction of GLUT1 mRNA by insulin but not by glucose deprivation. GLUT1 mRNA was decreased to basal levels within 12h following insulin withdrawal or glucose refeeding. Cycloheximide did not block this de-induction, suggesting that insulin and glucose deprivation do not increase GLUT1 mRNA expression by inhibiting the synthesis of ribonucleases. These findings indicate that insulin/IGF-1 increases both GLUT1 mRNA stability and transcription by both protein synthesis-dependent and independent mechanisms, whereas glucose deprivation enhances GLUT1 mRNA stability by mechanisms independent of de novo protein synthesis.

Animals↗
PubMed1990

Hexose specificity for downregulation of HepG2/brain-type glucose transporter gene expression in L6 myocytes.

Glucose deprivation of L6 myocytes results in the upregulation of glucose transporter activity, protein and mRNA. We have investigated the downregulation of transporter gene expression by glucose and other hexoses in glucose-deprived L6 myocytes. Glucose transport activity was measured as the uptake of 3H-2-deoxyglucose. Transporter protein and mRNA were detected by immunoblot and Northern blot analysis, respectively, with probes to the rat brain glucose transporter. Glucose deprivation of myocytes, in the absence and presence of insulin, increased 3H-2-deoxyglucose uptake, transporter protein and mRNA levels. Refeeding with glucose reversed the glucose deprivation effects on transport activity and mRNA within 12 h, with half-maximal effects at 1-2 mmol/l glucose. Mannose fully substituted for glucose. Refeeding with the non-metabolisable glucose analogues 2-deoxyglucose and 3-0-methylglucose, or with glucosamine or mannitol, downregulated 3H-2-deoxyglucose uptake but had little or no effect on transporter protein and mRNA expression. In contrast, glucose-6-phosphate markedly increased 3H-2-deoxyglucose uptake but partly downregulated transporter mRNA levels, whereas galactose had a small stimulatory effect on both 3H-2-deoxyglucose uptake and transporter mRNA; neither affected transporter protein levels. The transporter mRNA level was not affected by several metabolites (pyruvate, glyceraldehyde, glycerol) and amino acids (alanine, glutamine). These findings indicate that (i) there are independent pathways for hexose regulation of transport activity, protein and mRNA and (ii) down-regulation of transporter mRNA requires metabolism beyond hexose phosphate whereas glucose uptake may be regulated by direct interaction of hexoses with the transporter.

Animals↗
PubMed1989

Chronic stimulation of glucose transporter gene expression in L6 myocytes mediated via the insulin-like growth factor-1 receptor.

We have used differentiated L6 myocytes to investigate the regulation of glucose transporter gene expression by insulin and insulin-like growth factor-1 (IGF-1). Chronic exposure to insulin (1 microM) or IGF-1 (10 nm) resulted in a 2- to 5-fold stimulation of 3H-2-deoxy-D-glucose uptake and a corresponding increase in the expression of rat brain/HepG2-type glucose transporter mRNA (GTmRNA) and immunoreactive transporter protein. The dose responses to both insulin and IGF-1 for stimulation of glucose uptake were paralleled by the expression of GTmRNA. Glucose uptake and GTmRNA levels were half maximally stimulated by 350 and 100 nM insulin, respectively, or by 2 nM IGF-1. Comparison of receptor occupancy with stimulation of glucose uptake and GTmRNA expression suggests that insulin exerts its effects through the IGF-1 receptor. Fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, and phorbol ester had little or no effect on GTmRNA expression. These results demonstrate that the IGF-1 receptor mediates chronic regulation of transporter mRNA expression and protein synthesis and activity in cultured rat muscle cells.

Animals↗
PubMed1989

Insulin receptor expression in the Burkitt lymphoma cells Daudi and Raji.

The specific binding of insulin to either intact or Triton-solubilized Daudi cells (a Burkitt lymphoma cell line) was reduced by over 95% compared to that to control IM-9 lymphocytes due to a decrease in receptor number without a change in affinity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that 125I-labeled Daudi cells had reduced amounts (approximately 1/20th) of immunoprecipitable binding (alpha) subunit [mol wt (Mr), 130,000] of the receptor and a relatively abundant 210,000 Mr form not seen in IM-9 cells. The transmembranous (beta) subunit (Mr, 90,000) of the receptor, although not detected by 125I surface labeling, could be phosphorylated and, together with the 210,000 Mr form, exhibited the same 2-fold stimulation of phosphorylation by insulin as that in IM-9 cells. Northern blot hybridization revealed a decrease in Daudi cells of all four major species of insulin receptor mRNA. The Raji cell, another Burkitt lymphoma cell line, also exhibited reduced protein and genetic expression of the insulin receptor, indicating that reduced insulin receptor expression may be representative of other Burkitt lymphoma cell lines.

Blotting, Northern↗
PubMed1987

A novel whey protein synthesized only in late lactation by the mammary gland from the tammar (Macropus eugenii).

A major whey protein which appears in milk from the tammar wallaby (Macropus eugenii) only during the second half of lactation (late lactation protein-A, LLP-A) was purified to apparent homogeneity by ion-exchange chromatography and gel filtration. An Mr of 21,600 +/- 2000 was calculated from its amino acid composition. A computer-based comparison of the sequence of the first 69 amino acid residues with the Atlas of Protein Sequence data base showed no significant homology with known proteins. Antiserum to LLP-A was prepared in rabbits, and single radial immunodiffusion was used to measure the amounts of LLP-A in milk during the first 40 weeks of lactation. LLP-A was first detected at 26 weeks; thereafter its concentration increased abruptly, to reach a maximum of 26 g/l at approx. 36 weeks of lactation. Explants prepared from mammary gland biopsies at 20 and 35 weeks of lactation were exposed to [3H]amino acids for 8 h; immunoprecipitation of tissue extracts showed that, whereas the rate of casein synthesis was the same at both stages of lactation, LLP-A was synthesized only by the 35-week mammary gland.

Amino Acid Sequence↗
PubMed1987

Pituitary-induced lactation in mammary gland explants from the pregnant tammar (Macropus eugenii): a negative role for cyclic AMP.

1. alpha-Lactalbumin and casein have been isolated from tammar milk. 2. alpha-Lactalbumin was induced in mammary explants by culture with anterior pituitary. 3. Casein was induced maximally in the presence of a physiological concentration of prolactin alone. 4. Progesterone did not inhibit the prolactin-induced synthesis of casein, alpha-lactalbumin, galactosyltransferase or fatty acids. 5. Both dibutyryl cAMP and a combination of cholera toxin and IBMX did significantly inhibit the induction of casein and alpha-lactalbumin. 6. Progesterone withdrawal is not a component of the lactogenic trigger in this marsupial but cAMP may be a common intracellular signal for negative control of lactogenesis in both marsupials and eutherians.

Animals↗
PubMed1986

Glucagon and insulin regulate in vitro hepatic glycogenolysis in the axolotl Ambystoma mexicanum via changes in tissue cyclic AMP concentration.

Glucagon increases the rate of glycogenolysis in in vitro cultures of hepatic tissue from the axolotl Ambystoma mexicanum. The hormone causes an increase in the concentration of cyclic AMP in the tissue which is followed by activation of glycogen phosphorylase and subsequent breakdown of glycogen and release of glucose from the tissue. Insulin counteracts the glycogenolytic effect of glucagon by inhibiting the increase in tissue cyclic AMP concentration brought about by glucagon. This inhibitory effect of insulin is not seen in the presence of the phosphodiesterase inhibitor IBMX and so it appears that the initial action of insulin is a stimulation of cyclic AMP phosphodiesterase activity which lowers the tissue concentration of cyclic AMP and so counters the actions of hormones that act by raising the tissue concentration of cyclic AMP. This model for the mode of action of insulin is supported by the finding that insulin also interferes with the glycogenolytic actions of adrenaline, a second hormone which acts by raising tissue cyclic AMP concentrations.

1-Methyl-3-isobutylxanthine↗