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M A Magnuson

Publications and source records attributed to M A Magnuson.

At least 37 records · Page 2Linked to original sources
PubMed1999

Nuclear import of hepatic glucokinase depends upon glucokinase regulatory protein, whereas export is due to a nuclear export signal sequence in glucokinase.

Hepatic glucokinase (GK) moves between the nucleus and cytoplasm in response to metabolic alterations. Here, using heterologous cell systems, we have found that at least two different mechanisms are involved in the intracellular movement of GK. In the absence of the GK regulatory protein (GKRP) GK resides only in the cytoplasm. However, in the presence of GKRP, GK moves to the nucleus and resides there in association with this protein until changes in the metabolic milieu prompt its release. GK does not contain a nuclear localization signal sequence and does not enter the nucleus in a GKRP-independent manner because cells treated with leptomycin B, a specific inhibitor of leucine-rich NES-dependent nuclear export, do not accumulate GK in the nucleus. Instead, entry of GK into the nucleus appears to occur via a piggy-back mechanism that involves binding to GKRP. Nuclear export of GK, which occurs after its release from GKRP, is due to a leucine-rich nuclear export signal within the protein ((300)ELVRLVLLKLV(310)). Thus, GKRP appears to function as both a nuclear chaperone and metabolic sensor and is a critical component of a hepatic GK translocation cycle for regulating the activity of this enzyme in response to metabolic alterations.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Isolation and characterization of the mouse cytosolic phosphoenolpyruvate carboxykinase (GTP) gene: evidence for tissue-specific hypersensitive sites.

A 72 kilobase pair DNA fragment that contains the mouse phosphoenolpyruvate carboxykinase (PEPCK) gene locus, pck1, was isolated from a genomic bacterial artificial chromosome library. The region from approximately -5.5 to +6.6 kilobase pairs relative to the pck1 transcription start site was sequenced and exhibits a high degree of homology to the rat and human genes. Additionally, the chromatin structure of the PEPCK gene in mouse liver resembles that seen in rat. Backcross panel analysis of a microsatellite sequence confirms that the gene is located on chromosome 2. Hypersensitive site analysis was performed on nuclei isolated from the adipocyte cell line 3T3-F442A in the preadipose and adipose states. Several hypersensitive sites are present in the undifferentiated 3T3-F442A cells, before PEPCK mRNA is detected. The same sites are present after differentiation, however, the sensitivity of mHS 3 increases relative to the others. We conclude that the chromatin is open in 3T3-F442A cells and that factors are able to bind in the undifferentiated state but that something else is required for transcription.

3T3 Cells↗
PubMed1999

Tissue-specific knockout of the insulin receptor in pancreatic beta cells creates an insulin secretory defect similar to that in type 2 diabetes.

Dysfunction of the pancreatic beta cell is an important defect in the pathogenesis of type 2 diabetes, although its exact relationship to the insulin resistance is unclear. To determine whether insulin signaling has a functional role in the beta cell we have used the Cre-loxP system to specifically inactivate the insulin receptor gene in the beta cells. The resultant mice exhibit a selective loss of insulin secretion in response to glucose and a progressive impairment of glucose tolerance. These data indicate an important functional role for the insulin receptor in glucose sensing by the pancreatic beta cell and suggest that defects in insulin signaling at the level of the beta cell may contribute to the observed alterations in insulin secretion in type 2 diabetes.

Alleles↗
PubMed1999

Replacement by homologous recombination of the minK gene with lacZ reveals restriction of minK expression to the mouse cardiac conduction system.

The minK gene encodes a 129-amino acid peptide the expression of which modulates function of cardiac delayed rectifier currents (IKr and IKs), and mutations in minK are now recognized as one cause of the congenital long-QT syndrome. We have generated minK-deficient mice in which the bacterial lacZ gene has been substituted for the minK coding region such that beta-galactosidase expression is controlled by endogenous minK regulatory elements. In cardiac myocytes isolated from wild-type neonatal mice, IKs is rarely recorded, while IKr is common. In minK (-/-) myocytes, IKs is absent and IKr is significantly reduced and its deactivation slowed; these results further support a role for minK in modulating both IKs and IKr. Despite these changes, ECGs in (+/+) and (-/-) animals are no different at adult and at neonatal stages. ECG responses to isoproterenol are also similar in the 2 groups. beta-Galactosidase staining in postnatal minK (-/-) hearts is highly restricted, to the sinus-node region, caudal atrial septum, and proximal conducting system. Moreover, as early as embryonal day 11, segmentally restricted beta-galactosidase expression is observed in the portions of the sinoatrial and atrioventricular junctions that are thought to give rise to the conducting system, thereby implicating minK expression as an early event in conduction system development. More generally, the restricted nature of minK expression in the mouse heart suggests species-specific roles of this gene product in mediating the electrophysiological properties of the heart.

Animals↗
PubMed1999

Adenovirus-mediated knockout of a conditional glucokinase gene in isolated pancreatic islets reveals an essential role for proximal metabolic coupling events in glucose-stimulated insulin secretion.

The relationship between glucokinase (GK) and glucose-stimulated metabolism, and the potential for metabolic coupling between beta cells, was examined in isolated mouse islets by using a recombinant adenovirus that expresses Cre recombinase (AdenoCre) to inactivate a conditional GK gene allele (gklox). Analysis of AdenoCre-treated islets indicated that the gklox allele in approximately 30% of islet cells was converted to a nonexpressing variant (gkdel). This resulted in a heterogeneous population of beta cells where GK was absent in some cells. Quantitative two-photon excitation imaging of NAD(P)H autofluorescence was then used to measure glucose-stimulated metabolic responses of individual islet beta cells from gklox/lox mice. In AdenoCre-infected islets, approximately one-third of the beta cells showed markedly lower NAD(P)H responses. These cells also exhibited glucose dose responses consistent with the loss of GK. Glucose dose responses of the low-responding cells were not sigmoidal and reached a maximum at approximately 5 mM glucose. In contrast, the normal response cells showed a sigmoidal response with an KcatS0.5 of approximately 8 mM. These data provide direct evidence that GK is essential for glucose-stimulated metabolic responses in beta cells within intact islets and that intercellular coupling within the islet plays little or no role in glucose-stimulated metabolic responses.

Adenoviridae↗
PubMed1999

Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase.

Glucokinase (GK) gene mutations cause diabetes mellitus in both humans and mouse models, but the pathophysiological basis is only partially defined. We have used cre-loxP technology in combination with gene targeting to perform global, beta cell-, and hepatocyte-specific gene knock-outs of this enzyme in mice. Gene targeting was used to create a triple-loxed gk allele, which was converted by partial or total Cre-mediated recombination to a conditional allele lacking neomycin resistance, or to a null allele, respectively. beta cell- and hepatocyte-specific expression of Cre was achieved using transgenes that contain either insulin or albumin promoter/enhancer sequences. By intercrossing the transgenic mice that express Cre in a cell-specific manner with mice containing a conditional gk allele, we obtained animals with either a beta cell or hepatocyte-specific knock-out of GK. Animals either globally deficient in GK, or lacking GK just in beta cells, die within a few days of birth from severe diabetes. Mice that are heterozygous null for GK, either globally or just in the beta cell, survive but are moderately hyperglycemic. Mice that lack GK only in the liver are only mildly hyperglycemic but display pronounced defects in both glycogen synthesis and glucose turnover rates during a hyperglycemic clamp. Interestingly, hepatic GK knock-out mice also have impaired insulin secretion in response to glucose. These studies indicate that deficiencies in both beta cell and hepatic GK contribute to the hyperglycemia of MODY-2.

Albumins↗
PubMed1999

Mice lacking alpha-calcitonin gene-related peptide exhibit normal cardiovascular regulation and neuromuscular development.

alpha-Calcitonin gene-related peptide (alphaCGRP) is a pleiotropic peptide neuromodulator that is widely expressed throughout the Central and peripheral nervous systems. CGRP has been implicated in a variety of physiological processes including peripheral vasodilation, cardiac acceleration nicotinic acetylcholine receptor (AChR) synthesis and function, testicular descent, nociception, carbohydrate metabolism, gastrointestinal motility, neurogenic inflammation, and gastric acid secretion. To provide a better understanding of the physiological role(s) mediated by this peptide neurotransmitter, we have generated alphaCGRP-null mice by targeted modification in embryonic stem cells. Mice lacking alpha CGRP expression demonstrate no obvious phenotypic differences from their wild-type littermates. Detailed analysis of systemic cardiovascular function revealed no differences between control and mutant mice regarding heart rate and blood pressure under basal or exercise-induced conditions and subsequent to pharmacological manipulation. Characterization of neuromuscular junction in morphology including nicotinic receptor localization, terminal sprouting in response to denervation, developmental regulation of AChR subunit expression, and synapse elimination also revealed no differences in alphaCGRP-deficient animals. These results suggest that alphaCGRP is not required for the systemic regulation of cardiovascular hemodynamics or development of the neuromuscular junction.

Aging↗
PubMed1999

Mutants of glucokinase cause hypoglycaemia- and hyperglycaemia syndromes and their analysis illuminates fundamental quantitative concepts of glucose homeostasis.

AIMS/HYPOTHESIS: Mutations of the glucokinase gene cause hyperglycaemia or hypoglycaemia. A quantitative understanding of these defects of glucose homeostasis linked to the glucokinase gene was lacking. Therefore a database of kinetic variables of wild-type and 20 missense mutants of glucokinase was developed and used in mathematical modelling to predict the thresholds for glucose-stimulated insulin release. METHODS: Recombinant human glucokinase was generated in E. coli. The k(cat), glucose S(0.5), ATP K(m), and Hill number of glucokinase were determined. Inhibition by Stearoyl CoA and glucokinase regulatory protein and thermal stability were assayed for all mutants kinetically similar to wild-type glucokinase. A mathematical model predicting the threshold for glucose-stimulated insulin release was constructed. This model is based on the two substrate kinetics of glucokinase and the kinetic variables of the database. It is assumed that both glucokinase gene alleles are equally expressed in beta-cells and that induction of glucokinase occurs as a function of basal blood glucose. RESULTS: Large changes, varying greatly between mutants were found in nearly all variables. Glucokinase flux at threshold for glucose-stimulated insulin release was about 25 % of total phosphorylating potential in the normal beta-cell and this was used to predict thresholds for the mutant heterozygotes. Clinical data for maturity onset diabetes of the young type linked to the glucokinase gene and familial hyperinsulinaemic hypoglycaemia linked to the glucokinase gene and the glucokinase kinetic data of this study were used to test the model. The model predicts fasting blood glucose between 3 and 7 mmol/l in these cases. CONCLUSION/INTERPRETATION: A kinetics database of wild-type and 20 mutants of glucokinase was developed. Many kinetic differences were found for the mutants. The mathematical model to calculate the threshold for glucose-stimulated insulin release predicts fasting blood glucose between 3 and 7 mmol/l in subjects with glucokinase gene mutations. [Diabetologia 42: 1175-1186]

Adaptor Proteins, Signal Transducing↗
PubMed1999

Salt-sensitive hypertension and reduced fertility in mice lacking the prostaglandin EP2 receptor.

Prostaglandins (PGs) are ubiquitous lipid mediators derived from cyclooxygenase metabolism of arachidonic acid that exert a broad range of physiologic activities, including modulation of inflammation, ovulation and arterial blood pressure. PGE2, a chief cyclooxygenase product, modulates blood pressure and fertility, although the specific G protein-coupled receptors mediating these effects remain poorly defined. To evaluate the physiologic role of the PGE2 EP2 receptor subtype, we created mice with targeted disruption of this gene (EP2-/-). EP2-/- mice develop normally but produce small litters and have slightly elevated baseline systolic blood pressure. In EP2-/- mice, the characteristic hypotensive effect of intravenous PGE2 infusion was absent; PGE2 infusion instead produced hypertension. When fed a diet high in salt, the EP2-/- mice developed profound systolic hypertension, whereas wild-type mice showed no change in systolic blood pressure. Analysis of wild-type and EP2-/- mice on day 5 of pregnancy indicated that the reduced litter size of EP2-/- mice is due to a pre-implantation defect. This reduction of implanted embryos could be accounted for by impaired ovulation and dramatic reductions in fertilization observed on day 2 of pregnancy. These data demonstrate that the EP2 receptor mediates arterial dilatation, salt-sensitive hypertension, and also plays an essential part in female fertility.

Animals↗
PubMed1998

Targeted oncogenesis of hormone-negative pancreatic islet progenitor cells.

Transgenic mice containing an upstream glucokinase (betaGK) promoter- simian virus 40 T antigen (Tag) fusion gene develop neuroendocrine tumors primarily in the pancreas, gut, and pituitary. Pancreatic tumors from a line with delayed tumorigenesis were of two different types: insulinomas and noninsulinomas. The noninsulinomas are often periductal in location, express none of the four major islet peptide hormones, Glut-2, Pdx1, tyrosine hydroxylase, Pax4, Pax6, or Nkx6.1, but do express glucokinase, Sur1, Isl1, Hnf3beta, Hnf6, Beta2/NeuroD, and Nkx2.2. Cells from two different noninsulinoma tumors, when adapted to culture, began to express either insulin, glucagon, or somatostatin. Given the partial gene expression repertoire of the noninsulinoma tumors, their apparent periductal origin, and the ability of these cells to partially cytodifferentiate in culture, we suggest that these tumors are derived from islet progenitor cells. Thus, betaGK-Tag transgenic mice provide a new model system for studying the events that occur during both islet cell neogenesis and normal embryonic development.

Animals↗
PubMed1998

Dopamine biosynthesis is selectively abolished in substantia nigra/ventral tegmental area but not in hypothalamic neurons in mice with targeted disruption of the Nurr1 gene.

To ascertain the function of an orphan nuclear receptor Nurr1, a transcription factor belonging to a large gene family that includes receptors for steroids, retinoids, and thyroid hormone, we generated Nurr1-null mice by homologous recombination. Mice, heterozygous for a single mutated Nurr1 allele, appear normal, whereas mice homozygous for the null allele die within 24 h after birth. Dopamine (DA) was absent in the substantia nigra (SN) and ventral tegmental area (VTA) of Nurr1-null mice, consistent with absent tyrosine hydroxylase (TH), L-aromatic amino acid decarboxylase, and other DA neuron markers. TH immunoreactivity and mRNA expression in hypothalamic, olfactory, and lower brain stem regions were unaffected. L-Dihydroxyphenylalanine treatments, whether given to the pregnant dams or to the newborns, failed to rescue the Nurr1-null mice. We were unable to discern differences between null and wild-type mice in the cellularity, presence of neurons, or axonal projections to the SN and VTA. These findings provide evidence for a new mechanism of DA depletion in vivo and suggest a unique role for Nurr1 in fetal development and/or postnatal survival.

Animals↗
PubMed1998

Pancreatic beta-cell glucokinase: closing the gap between theoretical concepts and experimental realities.

There remains a wide gap between theoretical concepts and experimental realities in the enzyme kinetics and biochemical genetics of the pancreatic beta-cell glucokinase-glucose sensor. It is the goal of present efforts in many laboratories to bridge this gap. This perspective intends to provide a timely review of this crucial aspect of research in glucose homeostasis. It deals briefly with some fundamentals of glucokinase enzyme kinetics, offers some pertinent biochemical genetic considerations, takes stock of the current experimental database of the field by emphasizing human studies and referring to recent mouse studies, and ventures a few extrapolations into the future of this endeavor.

Animals↗
PubMed1998

Activation of glycine and glutamate receptors increases intracellular calcium in cells derived from the endocrine pancreas.

We studied calcium signaling in a newly described pancreatic cell line, GK-P3, that expresses functional amino acid neurotransmitter receptors. GK-P3 cells express the first strychnine-sensitive glycine receptors reported in a permanent cell line. In addition, GK-P3 cells express alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors. Both types of amino acid receptors showed electrophysiological and pharmacological behavior similar to their neuronal counterparts. The glycine receptors were permeable to Cl- and blocked by the selective antagonist strychnine. AMPA receptors showed limited permeability to Ca2+, were blocked by 6-cyano-2, 3-dihydroxy-7-nitroquinoxaline, and were potentiated by cyclothiazide. Interestingly, activation of either receptor type increased intracellular Ca2+ measured by digital imaging of Fura-2 fluorescence. These Ca2+ signals were completely blocked by 30 microM La3+, suggesting that the Ca2+ entered the cells largely through voltage-dependent Ca2+ channels. Alterations in the extracellular concentrations of Cl- and/or HCO3- had only marginal effects on glycine-evoked Ca2+ signals. However, increases in intracellular Ca2+ mediated by AMPA receptors were absent when the extracellular Na+ was replaced with an impermeant cation, N-methyl-D-glucamine. We conclude that activation of ligand-gated cation or anion channels depolarize GK-P3 cells sufficiently to activate their voltage-gated Ca2+ channels leading to increases in intracellular Ca2+ concentration. Thus, glycine and glutamate receptors may regulate Ca2+-dependent secretory mechanisms in islet cells by altering the membrane potential of these cells. Our data in GK-P3 cells support the growing weight of evidence for a role of amino acid neurotransmitters in pancreatic islets and introduce strychnine-sensitive glycine receptors as a novel target of amino acid neurotransmitter regulation in islets.

Animals↗
PubMed1997

Variable expression of hepatic glucokinase in mice is due to a regulational locus that cosegregates with the glucokinase gene.

The Gk activity locus affects expression of hepatic glucokinase (GK) in mice. Analysis of microsatellites within the mouse GK gene locus revealed two major haplotypes in 19 of 22 inbred strains predictive of either high or low hepatic GK gene expression. C3H/HeJ mice, a high-activity strain, and two other wild-derived strains contain less common haplotypes. No coding sequence differences were found in hepatic GK-coding sequences from representative high and low Gk activity strains, thereby excluding kinetic abnormalities as the basis for hepatic GK activity differences. Screening of approximately 10 kb of potential regulatory DNA, including all eight known and three of four newly identified DNase I-hypersensitive sites, by restriction enzyme fingerprinting-single-strand conformation analysis revealed a tetranucleotide microsatellite, the length of which was also predictive of the Gk activity phenotype. This tetranucleotide repeat is located in the first intron of the hepatic transcription unit and lies close to a newly identified liver-specific DNase I-hypersensitive site. These results indicate that the Gk activity alleles are a regulational locus associated with the GK gene locus.

Animals↗
PubMed1997

Cell-specific expression and regulation of a glucokinase gene locus transgene.

Transgenic mice containing one or more extra copies of the entire glucokinase (GK) gene locus were generated and characterized. The GK transgene, an 83-kilobase pair mouse genomic DNA fragment containing both promoter regions, was expressed and regulated in a cell-specific manner, and rescued GK null lethality when crossed into mice bearing a targeted mutation of the endogenous GK gene. Livers from the transgenic mice had elevated GK mRNA, protein, and activity levels, compared with controls, and the transgene was regulated in liver by dietary manipulations. The amount of GK immunoreactivity in hepatocyte nuclei, where GK binds to the GK regulatory protein, was also increased. Pancreatic islets displayed increased GK immunoreactivity and NAD(P)H responses to glucose, but only when isolated and cultured in 20 mM glucose, as a result of the hypoglycemic phenotype of these mice (Niswender, K. D., Shiota, M., Postic, C., Cherrington, A. D., and Magnuson, M. A. (1997) J. Biol. Chem. 272, 22604-22609). Together, these results indicate that the region of the gene from -55 to +28 kilobase pairs (relative to the liver GK transcription start site) contains all the regulatory sequences necessary for expression of both GK isoforms, thereby placing an upper limit on the size of the GK gene locus.

Animals↗
PubMed1997

Effects of increased glucokinase gene copy number on glucose homeostasis and hepatic glucose metabolism.

The relationship between glucokinase (GK) gene copy number and glucose homeostasis was studied in transgenic mice with additional copies of the entire GK gene locus (Niswender, K. D., Postic, C., Jetton, T. L., Bennett, B. D., Piston, D. W., Efrat, S., and Magnuson, M. A. (1997) J. Biol. Chem. 272, 22564-22569). The plasma glucose concentration was reduced by 25 +/- 3% and 37 +/- 4% in mice with one or two extra copies of the gene locus, respectively. The basis for the hypoglycemic phenotype was determined using metabolic tracer techniques in chronically cannulated, conscious mice with one extra GK gene copy. Under basal conditions (6-h fasted) transgenic mice had a lower blood glucose concentration (-12 +/- 1%) and a slightly higher glucose turnover rate (+8 +/- 3%), resulting in a significantly higher glucose clearance rate (+21 +/- 2%). Plasma insulin levels were not different, suggesting that increased glucose clearance was due to augmented hepatic, not islet, GK gene expression. Under hyperglycemic clamp conditions the transgenic mice had glucose turnover and clearance rates similar to the controls, but showed a lower plasma insulin response (-48 +/- 5%). Net hepatic glycogen synthesis was markedly elevated (+360%), whereas skeletal muscle glycogen synthesis was decreased (-40%). These results indicate that increased GK gene dosage leads to increased hepatic glucose metabolism and, consequently, a lower plasma glucose concentration. Increased insulin secretion was not observed, even though the transgene is expressed in islets, because hypoglycemia causes a down-regulation in islet GK content (Niswender, K. D., Postic, C., Jetton, T. L., Bennett, B. D., Piston, D. W., Efrat, S., and Magnuson, M. A. (1997) J. Biol. Chem. 272, in press).

Animals↗
PubMed1997

Essential role of the tyrosine kinase substrate phospholipase C-gamma1 in mammalian growth and development.

The activation of many tyrosine kinases leads to the phosphorylation and activation of phospholipase C-gamma1 (PLC-gamma1). To examine the biological function of this protein, homologous recombination has been used to selectively disrupt the Plcg1 gene in mice. Homozygous disruption of Plcg1 results in embryonic lethality at approximately embryonic day (E) 9.0. Histological analysis indicates that Plcg1 (-/-) embryos appear normal at E 8.5 but fail to continue normal development and growth beyond E 8.5-E9.0. These results clearly demonstrate that PLC-gamma1 with, by inference, its capacity to mobilize second messenger molecules is an essential signal transducing molecule whose absence is not compensated by other signaling pathways or other genes encoding PLC isozymes.

Animals↗