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F M Matschinsky

Publications and source records attributed to F M Matschinsky.

At least 37 records · Page 2Linked to original sources
PubMed1998

Mechanisms of spontaneous cytosolic Ca2+ transients in differentiated human neuronal cells.

We have studied Ca2+ homeostasis in a unique model of human neurons, the NT2N cell, which differentiates from a human teratocarcinoma cell line, NTera2/C1.D1 by retinoic acid treatment. When perifused with Krebs-HEPES buffer containing 2.5 mM CaCl2, fura-2 loaded NT2N cells produced spontaneous cytosolic Ca2+ oscillations, or Ca2+ transients. These cytosolic Ca2+ transients were not blocked by antagonists of glutamate (6-cyano-7-nitroquinoxaline-2,3-dione and D(-)-2-amino-5-phosphonopentanoic acid) or muscarinic (atropine) receptors. Omission of extracellular Ca2+ completely abolished Ca2+ oscillations and decreased the average Ca2+ level from 106 +/- 14 nM to 59 +/- 8 nM. Addition of the L-type Ca2+ channel blocker nifedipine (1 or 10 microM) or of the N-type inhibitor omega-conotoxin GVIA (5 microM) significantly, although incompletely, suppressed Ca2+ oscillations, while omega-conotoxin MVIIC (5 microM), a selective antagonist of P- and Q-channels, had no effect. Ni2+, at 100 microM, a concentration selective for T-type channels, did not inhibit Ca2+ transients. Non-specific blockage of Ca2+ channels by higher concentrations of Ni2+ (2-5 mM) or Co2+ (1 mM) abolished Ca2+ oscillations completely. The endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin (1 microM), slightly decreased Ca2+ oscillation frequency, and induced a small transitory increase in the average cytosolic Ca2+ concentration. The mRNAs of L- (alpha1D subunit) and N-type (alpha1B subunit) Ca2+ channel were present in NT2N cells, while that of a T-type Ca2+ channel (alpha1-subunit) was not present in the NT2N cells as shown by reverse transcription-polymerase chain reaction. In conclusion, NT2N neuronal cells generate cytosolic Ca2+ oscillations mainly by influx of extracellular Ca2+ through multiple channels, which include L- and N-type channels, and do not require activation of glutamate or muscarinic receptors.

Calcium↗
PubMed1998

Cytotoxic effect of beta-amyloid on a human differentiated neuron is not mediated by cytoplasmic Ca2+ accumulation.

The effects of synthetic beta-amyloid (A beta1-42) on cell viability and cellular Ca2+ homeostasis have been studied in the human neuron-like NT2N cell, which differentiates from a teratocarcinoma cell line, NTera2/C1.D1, by retinoic acid treatment. NT2N viability was measured using morphological criteria and fluorescent live/dead staining and quantified using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide metabolism. A beta1-42 dose-dependently caused NT2N cell death when it was present in the cell culture for 14 days but had no effect on viability when it was present for 4 days. The lowest effective concentration was 4 microM, and the strongest effect was produced by 40 microM. Control NT2N cells produced spontaneous cytosolic Ca2+ oscillations under basal conditions. These oscillations were inhibited dose-dependently (0.4-40 microM) by A beta1-42 that was present in the cell culture for 1 or 4 days. Ca2+ wave frequency was decreased from 0.21 +/- 0.02 to 0.05 +/- 0.02/min, amplitude from 88 +/- 8 to 13 +/- 4 nM, and average Ca2+ level from 130 +/- 8 to 58 +/- 3 nM. The Ca2+ responses to 30 mM K+ and 100 microM glutamate were not different between control and A beta-treated cells. Thus, the results do not support the hypothesis that cytosolic early Ca2+ accumulation mediates A beta-induced NT2N cell death.

Amyloid beta-Peptides↗
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↗
PubMed1997

Regulation of insulin preRNA splicing by glucose.

Glucose tightly regulates the synthesis and secretion of insulin by beta cells in the pancreatic islets of Langerhans. To investigate whether glucose regulates insulin synthesis at the level of insulin RNA splicing, we developed a method to detect and quantify a small amount of RNA by using the branched DNA (bDNA) signal-amplification technique. This assay is both sensitive and highly specific: mouse insulin II mRNA can be detected from a single beta cell (betaTC3 cells or mouse islets), whereas 1 million non-insulin-producing alpha cells (alphaTC1.6 cells) give no signal. By using intron and exon sequences, oligonucleotide probes were designed to distinguish the various unspliced and partially spliced insulin preRNAs from mature insulin mRNA. Insulin RNA splicing rates were estimated from the rate of disappearance of insulin preRNA signal from beta cells treated with actinomycin D to block transcription. We found that the two introns in mouse insulin II are not spliced with the same efficiency. Intron 2 is spliced out more efficiently than intron 1. As a result, some mRNA retaining intron 1 enters the cytoplasm, making up approximately 2-10% of insulin mRNA in the cell. This partially spliced cytoplasmic mRNA is quite stable, with a half-life similar to the completely spliced form. When islets grown in high glucose are shifted to low glucose medium, the level of insulin preRNA and the rate of splicing fall significantly. We conclude that glucose stimulates insulin gene transcription and insulin preRNA splicing. Previous estimates of insulin transcription rates based on insulin preRNA levels that did not consider the rate of splicing may have underestimated the effect of glucose on insulin gene transcription.

Animals↗
PubMed1997

Structural instability of mutant beta-cell glucokinase: implications for the molecular pathogenesis of maturity-onset diabetes of the young (type-2).

The catalytic function and thermal stability of wild-type and mutant recombinant human pancreatic beta-cell glucokinase was investigated. The mutants E70K and E300K, which are thought to be the cause of impaired insulin production by the pancreatic beta-cell and decreased glucose uptake by the liver of patients with maturity-onset diabetes of the young, were found to be functionally indistinguishable from the wild-type, i.e. their kcat.S0.5, inflection point and h were normal. However, these two mutants showed markedly reduced stability under a variety of test conditions. Glucokinase instability, not low enzyme catalytic activity, may be the cause of diabetes mellitus with E70K and E300K mutants.

Diabetes Mellitus, Type 2↗
PubMed1997

Chronic effect of fatty acids on insulin release is not through the alteration of glucose metabolism in a pancreatic beta-cell line (beta HC9).

Hyperinsulinaemia in the fasting state and a blunted insulin secretory response to acute glucose stimulation are commonly observed in obesity associated non-insulin-dependent diabetes mellitus. Hyperlipidaemia is a hallmark of obesity and may play a role in the pathogenesis of this beta-cell dysfunction because glucose metabolism in pancreatic beta cells may be altered by the increased lipid load. We tested this hypothesis by assessing the chronic effect of oleic acid on glucose metabolism and its relationship with glucose-induced insulin release in beta HC9 cells in tissue culture. Our results show: (1) A 4-day treatment with oleic acid caused an enhancement of insulin release at 0-5 mmol/l glucose concentrations while a significant decrease in insulin release occurred when the glucose level was greater than 15 nmol/l; (2) Hexokinase activity was increased and a corresponding left shift of the dose-dependency curve of glucose usage was observed associated with inhibition of glucose oxidation in oleic acid treated beta HC9 cells, yet the presumed glucose-related ATP generation did not parallel the change in insulin release due to glucose; (3) The rate of cellular respiration was markedly increased in oleic acid treated beta HC9 cells both in the absence of glucose and at all glucose concentrations tested. This enhanced oxidative metabolism may explain the increased insulin release at a low glucose level but is clearly dissociated from the blunted insulin secretion at high glucose concentrations. We conclude that a reduction of oxidative metabolism in pancreatic beta cells is unlikely to be the cause of the dramatic effect that high levels of non-esterified fatty acids have on glucose-induced insulin release.

Animals↗
PubMed1997

GLUT1 is adequate for glucose uptake in GLUT2-deficient insulin-releasing beta-cells.

GLUT2 may play an important role in pancreatic beta-cell glucose metabolism. A decrease in glucose uptake due to underexpression of GLUT2 has been considered as the cause of beta-cell dysfunction in diabetes with different pathogenesis. However, this view has been challenged by recent studies, in which the underexpression of GLUT2 was not accompanied by a decrease in glucose uptake. Our present aim is to evaluate the presumed importance of GLUT2 in maintaining the efficiency of beta-cell glucose uptake. We studied the kinetic characteristics of 3-O-methylglucose uptake in two beta-cell lines. One of these is the beta TC3 cell line which expresses GLUT1 and the other is the beta HC9 cell line which expresses both GLUT1 and GLUT2. Under equilibrium exchange conditions, 3-O-methylglucose transport in these two cell lines showed similar values of K(m) and V(max). The apparent IC50 of cytochalasin B for inhibiting 3-O-methylglucose transport in beta HC9 cells was nine times as high as in beta TC3 cells, indicating that GLUT1 is the critically important glucose transporter in the beta TC3 cell line and GLUT2 in the beta HC9 cell line. In both cell lines, the rates of glucose uptake were at least three times as fast as that of glucose phosphorylation. Our results suggest that GLUT1 is able to compensate for GLUT2 loss as it occurs in beta TC3 and maintains a commensurately high capacity of glucose uptake to sustain glucose metabolism in pancreatic beta-cells.

3-O-Methylglucose↗
PubMed1997

Measurement and modeling of glucose-6-phosphatase in pancreatic islets.

In the beta-cells of the pancreas, glucose phosphorylation carried out by glucokinase is the rate-controlling step in glycolysis, and the kinetic characteristics of glucokinase govern to a high degree the dose-response relationship between glucose and insulin release. Because glucose-6-phosphatase (G-6-Pase) opposes the action of glucokinase, it may have a regulatory role in the release of insulin in response to glucose if the enzyme is present in the beta-cells. A number of researchers have reported finding high levels of G-6-Pase in islets, but quantitation of its activity remains controversial, mainly because of difficulties in solubilizing a particulate enzyme. Therefore a method developed to measure functional glucose phosphorylation activity in intact brain was applied (Chi, M. M.-Y., M. E. Pusateri, J. G. Carter, B. J. Norris, D. B. McDougal, Jr., and O. H. Lowry. Anal. Biochem. 161: 508-513, 1987), and the rates of accumulation and disappearance of 2-deoxyglucose 6-phosphate (DG-6-P) in freshly harvested islets were determined as a measure of glucose cycling. Islets were incubated in the presence of 30 mM 2-deoxyglucose (DG) for 60 min, and subsequently the incubation medium was replaced with medium containing no DG, but instead high levels of mannoheptulose as a blocker of phosphorylation. The content of DG-6-P in the islets was measured at strategic times during the protocol. As predicted by a mathematical model, DG-6-P accumulated in the presence of DG and decayed after its washout. Both of these results are consistent with islets containing dephosphorylation activity for this substrate. The kinetic curves were fit using a mathematical model, and the maximal G-6-Pase activity was estimated to be 0.13 +/- 0.005 micromol x g(-1) x min(-1). However, when the physiological effect of this amount of G-6-Pase activity was assessed by use of a model of glycolysis, it was found that the impact on glucose cycling and usage was insignificant. It was concluded that normal islets do contain measurable activity for dephosphorylating glucose 6-phosphate but that this enzymatic reaction does not play a role in glucose metabolism and sensing by the normal beta-cell.

Animals↗
PubMed1996

Glucose metabolism and insulin release in mouse beta HC9 cells, as model for wild-type pancreatic beta-cells.

Glucose metabolism and its relationship with glucose-induced insulin release were studied in beta HC9 and beta TC3 cells to identify and characterize key factors controlling the intermediary metabolism of glucose and glucose-induced insulin release. The beta HC9 cell line, derived from pancreatic islets with beta-cell hyperplasia, is characterized by a normal concentration-dependency curve for glucose-stimulated insulin release, whereas the beta TC3 cell line, derived from pancreatic beta-cell tumors, shows a marked leftward shift of this curve. Maximum velocity and the Michaelis-Menten constant of glucose uptake in beta HC9 and beta TC3 cells were similar, even though GLUT-2 expression in these two cell lines differed. In both cell lines, the kinetic characteristics of glucose usage, glucose oxidation, and glucose-induced oxygen consumption were similar to those of glucose phosphorylation, indicating that the kinetics of glucose metabolism from the glucose phosphorylation step in the cytosol to the mitochondrial process of oxidative phosphorylation are determined by the glucose-phosphorylating enzyme, that is, by glucokinase in beta HC9 cells and by hexokinase in beta TC3 cells. Thus beta HC9 cells provide an opportunity for the quantitative analysis of glucose metabolism, the associated generation of coupling factors, and other essential beta-cell functions involved in glucose sensing and insulin secretion.

Animals↗
PubMed1996

Effect of a glucokinase inhibitor on energy production and insulin release in pancreatic islets.

Glucokinase has exclusively high control strength on glucose usage in the pancreatic beta-cell. However, glucokinase also has extraordinarily high control strength on insulin secretion, which is linked to the phosphate potential, [ATP]/([ADP][Pi]) (F.M. Matschinsky, Y.Liang, P. Kesavan, L. Wang, P. Froguel, G. Velho, D. Cohen, M.A. Permutt, Y. Tanizawa, T.L. Jetton, K. Niswender, and M.A. Magnuson. J. Clin. Invest. 92: 2092-2098, 1993). We propose that the ATP produced via the tricarboxylic acid cycle is approximately constant, irrespective of the glucose level. Furthermore, the component of ATP production that is derived from glycolysis and glycolytically derived NADH, which is shuttled into the mitochondria, is a critical signal controlling the ionic events leading to insulin secretion, as suggested previously (M. J. MacDonald. Diabetes 39: 1461-1466, 1990 and I.D. Dukes, M.S. McIntyre, R.J. Mertz, L.H. Philipson, M.W. Roe, B. Spencer, and J.F. Worley III. J. Biol. Chem. 269: 10979-10982, 1994). To test this hypothesis, glucose usage, oxidation, and insulin secretion were measured in cultured rat islets over a wide range of concentrations of glucose and mannoheptulose, an inhibitor of glucokinase. These data were fit to a mathematical model that predicts that glucokinase will govern the rate of glucose usage and ATP production and will also have a strong, but not complete, control over the rate of glucose oxidation, the phosphate potential, and insulin release. Mannoheptulose caused an inhibition of all three fluxes. The estimates of the mechanistic parameters of the model [maximal velocity (Vmax) and Michaelis constant for glucokinase, Vmax for hexokinase and glucose transport, and the inhibition constant of mannoheptulose to glucokinase] were similar to those obtained in vitro. Thus the data are consistent with a model in which the primary importance of glycolysis in transducing the glucose signal into changes of the phosphate potential imparts to glucokinase a high control strength on glucose-induced insulin secretion.

Animals↗
PubMed1996

Banting Lecture 1995. A lesson in metabolic regulation inspired by the glucokinase glucose sensor paradigm.

Special features of glucose metabolism in pancreatic beta-cells are central to an understanding of the physiological role of these cells in glucose homeostasis. Several of these characteristics are emphasized: a high-capacity system for glucose transport; glucose phosphorylation by the high-Km glucokinase (GK), which is rate-limiting for glucose metabolism and determines physiologically the glucose dependency curves of many processes in beta-cell intermediary and energy metabolism and of insulin release and is therefore viewed as glucose sensor; remarkably low activity of lactate dehydrogenase and the presence of effective hydrogen shuttles to allow virtually quantitative oxidation of glycolytic NADH; the near absence of glycogen and fatty acid synthesis and of gluconeogenesis, such that intermediary metabolism is primarily catabolic; a crucial role of mitochondrial processes, including the citric acid cycle, electron transport, and oxidative phosphorylation with FoF1 ATPase governing the glucose-dependent increase of the ATP mass-action ratio; a Ca(2+)-independent glucose-induced respiratory burst and increased ATP production in beta-cells as striking manifestations of crucial mitochondrial reactions; control of the membrane potential by the mass-action ratio of ATP and voltage-dependent Ca2+ influx as signal for insulin release; accumulation of malonyl-CoA, acyl-CoA, and diacylglycerol as essential or auxiliary metabolic coupling factors; and amplification of the adenine nucleotide, lipid-related, and Ca2+ signals to recruit many auxiliary processes to maximize insulin biosynthesis and release. The biochemical design also suggests certain candidate diabetes genes related to fuel metabolism: low-activity and low-stability GK mutants that explain in part the maturity-onset diabetes of the young (MODY) phenotype in humans and mitochondrial DNA mutations of FoF1 ATPase components thought to cause late-onset diabetes in BHEcdb rats. These two examples are chosen to illustrate that metabolic reactions with high control strength participating in beta-cell energy metabolism and generating coupling factors and intracellular signals are steps with great susceptibility to genetic, environmental, and pharmacological influences. Glucose metabolism of beta-cells also controls, in addition to insulin secretion and insulin biosynthesis, an adaptive response to excessive fuel loads and may increase the beta-cell mass by hypertrophy, hyperplasia, and neogenesis. It is probable that this adaptive response is compromised in diabetes because of the GK or ATPase mutants that are highlighted here. A comprehensive knowledge of beta-cell intermediary and energy metabolism is therefore the foundation for understanding the role of these cells in fuel homeostasis and in the pathogenesis of the most prevalent metabolic disease, diabetes.

Adenine Nucleotides↗
PubMed1996

Wortmannin inhibits insulin secretion in pancreatic islets and beta-TC3 cells independent of its inhibition of phosphatidylinositol 3-kinase.

Glucose is the primary stimulus for insulin secretion by pancreatic beta-cells, and it triggers membrane depolarization and influx of extracellular Ca2+. Cholinergic agonists amplify insulin release by several pathways, including activation of phospholipase C, which hydrolyzes membrane polyphosphoinositides. A novel phospholipid, phosphatidylinositol 3,4,5- trisphosphate [PtdIns(3,4,5)P3], a product of phosphatidylinositol 3-kinase (PI 3-kinase), has recently been found in various cell types. We demonstrate by immunoblotting that PI 3-kinase is present in both cytosolic and membrane fractions of insulin-secreting beta-TC3 cells and in rat islets. The catalytic activity of PI 3-kinase in immunoprecipitates of islets and beta-TC3 cells was measured by the production of radioactive phosphatidylinositol 3-monophosphate from phosphatidylinositol (PtdIns) in the presence of [gamma-32P]ATP. Wortmannin, a fungal metabolite, dose dependently inhibited PI 3-kinase activity of both islets and beta-TC3 cells, with an IC50 of 1 nmol/l and a maximally effective concentration of 100 nmol/l, when it was added directly to the kinase assay. However, if intact islets were incubated with wortmannin and PI 3-kinase subsequently was determined in islet immunoprecipitates, approximately 50% inhibition of PI 3-kinase activity (but no inhibition of glucose- and carbachol-stimulated insulin secretion) from intact islets was obtained at wortmannin concentrations of 100 nmol/l. Wortmannin, at higher concentrations (1 and 10 micromol/l), inhibited glucose- and carbachol-induced insulin secretion of Intact rat islets by 58 and 92%, respectively. Wortmannin had no effect on the basal insulin release from rat islets. A similar dose curve of inhibition of glucose- and carbachol-induced insulin secretion by wortmannin was obtained when beta-TC3 cells were used. Cellular metabolism was, not changed by any wortmannin concentrations tested (0.01-10 micromol/l). Both basal cytosolic [Ca2+]i and carbamyl choline-induced increases of [Ca2]i were unaffected by wortmannin in the presence of 2.5 mmol/l Ca2+, while Ca2+ mobilization from intracellular stores was partially decreased by wortmannin. Together, these data suggest that wortmannin at concentrations that inhibit PI 3-kinase does not affect insulin secretion. PI 3-kinase is unlikely to have a major role in insulin secretion induced by glucose and carbachol.

Androstadienes↗
PubMed1995

Variable effects of maturity-onset-diabetes-of-youth (MODY)-associated glucokinase mutations on substrate interactions and stability of the enzyme.

Mutations in the human glucokinase (GK) gene are thought to cause maturity-onset diabetes of youth (MODY) by leading to the production of enzymes with reduced catalytic activities and increased glucose Km values. However, in some cases the diabetic phenotype is more severe than might be predicted from these apparent kinetic effects alone. To determine whether these mutations might also effect other characteristics of the enzyme, nine MODY-associated mutants were expressed as fusion proteins with Schistosoma japonicum glutathione S-transferase (GST) and compared with three wild-type human GK isoforms that were also expressed in the same manner. Three GST-GK isoforms (liver 1, liver 2 and islet) were kinetically indistinguishable from each other and from purified rat liver GK. Noteworthy is a glucose-induced fit effect for the interaction of trinitrophenyl (TNP)-ATP with GST-GK, whereby glucose significantly increased the affinity of TNP-ATP binding to GST-GK without changing the stoichiometry of binding. The nine MODY-associated mutations studied either showed diminished catalytic activity, substrate affinities, allosteric regulation, or stability of the fusion enzyme. We conclude that: (1) Gly261 and Lys414 are important for ATP binding; (2) Val203 may be essential for a glucose-induced fit effect; and (3) the stability of fusion protein may be significantly reduced when Glu300 is replaced by Lys. These results suggest that, in addition to effects on the Km and Vmax. of GK, a decrease in the ATP-binding affinity or stability of the mutated enzyme may also contribute to a reduction of GK activity in individuals with GK-MODY. In the B-cell this would have the effect of blunting glucose-stimulated insulin release, thereby contributing to the diabetic phenotype.

Adenosine Triphosphate↗
PubMed1995

Mouse embryonic stem cells express receptors of the insulin family of growth factors.

Insulin and insulin-like growth factors (IGF-I and -II) are members of a family of growth factors which are known to be developmentally regulated during preimplantation mouse embryogenesis. The physiological actions of the insulin family of growth factors are mediated by interactions with specific cell surface receptors that are detectable on the cells of preimplantation mouse embryos. Mouse embryonic stem (ES) cells are totipotent cells derived directly from the inner cell mass of the blastocyst. ES cells have the ability to differentiate into all three germ layers and have unlimited growth potential under certain culture conditions. The great advantage of ES cells is the ability to obtain large amounts of tissue for biochemical studies as compared with preimplantation embryos. To examine in greater detail the biological actions of the insulin family of growth factors, the expression of their cognate receptors on ES cells was examined. ES cells were cultured in DMEM medium supplemented with leukemia inhibitory factor (LIF) to maintain the undifferentiated state. Receptor expression was evaluated at the mRNA level using the reverse transcription polymerase chain reaction (RT-PCR), and at the protein level by radioactive labeled ligand-receptor binding assay. Using RT-PCR, mRNAs of all three growth factor receptors were detected in ES cells. Messenger RNA from ES cells was reverse transcribed into cDNA by AMV reverse transcriptase at 42 degrees C for 1 hr. The reverse transcription reaction was amplified with Taq polymerase and specific primers for insulin, IGF-I, or IGF-II receptors by PCR. RT-PCR and the control plasmid cDNA PCR products were resolved electrophoretically on 3% agarose gels. Each amplified PCR product showed the predicted correct size.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Mathematical model of beta-cell glucose metabolism and insulin release. I. Glucokinase as glucosensor hypothesis.

To quantitatively test the theory that glucokinase controls the rate of glucose metabolism and therefore the rate of insulin secretion, a minimal mathematical model of glycolysis in the pancreatic beta-cell was developed. The model represents our current hypothesis of how the normal beta-cell transduces the glucose signal. In this report, the model was used to address questions regarding the control strength of transport, hexokinase, glucose-6-phosphatase, and phosphofructokinase in the metabolism of glucose. The hypothesis that fructose 6-phosphate and a protein regulator modulate glucokinase activity was evaluated by simulation analysis, as was the possibility that glucose-6-phosphatase, working in concert with phosphofructokinase, can modulate the glucose-sensing system. It was found that, in the absence of glucose-6-phosphatase, transport, hexokinase, and phosphofructokinase do not greatly influence the rate of glucose metabolism unless their activities are dramatically altered from the measured values. Glucose metabolism was profoundly affected by the activity of glucokinase. However, in the presence of glucose-6-phosphatase, the ratio of glucose-6-phosphatase to phosphofructokinase activities was a very important parameter, and this potential control mechanism deserves more attention. The results further support the notion that glucokinase is indeed the glucosensor of the beta-cell and that modeling the system in toto provides quantitative evaluation needed to interpret the experimental tests of hypotheses.

Animals↗
PubMed1994

Analysis of upstream glucokinase promoter activity in transgenic mice and identification of glucokinase in rare neuroendocrine cells in the brain and gut.

A transgene consisting of an upstream glucokinase (GK) promoter fragment linked to coding sequences of the human growth hormone gene was expressed in certain neuroendocrine cells of the pancreas, pituitary, brain, gut, thyroid, and lungs of mice. In pancreas, the transgene was expressed in a nonuniform manner among beta cells and in a variable but substantial fraction of the other islet cell types. In pituitary, it was expressed in corticotropes, and in brain, it was expressed in cells of the medial hypothalamus. Within the gut transgene expression was detected in a subset of enteroendocrine cells of the stomach and duodenal epithelium, some of which also exhibited glucagon-like polypeptide-1 immunoreactivity. In thyroid, transgene expression was observed in C cells of neonatal animals, whereas in the lung, it was expressed among rare endocrine cells of the bronchopulmonary mucosa. RNA polymerase chain reaction analysis of human growth hormone mRNA corroborated the tissue-specific transgene expression pattern. Prompted by the finding of transgene expression in specific neuroendocrine cells, we sought to determine whether GK mRNA and GK itself was also expressed in the brain and gut, tissues not previously associated with the expression of this enzyme. Using rat tissues, GK mRNA was detected by RNA polymerase chain reaction in both the brain and intestine and was localized to specific cells in the hypothalamus and enteric mucosa by in situ hybridization. A high Km glucose phosphorylating activity was detected from isolated rat jejunal enterocytes that displayed a chromatographic elution profile identical to hepatic GK. GK immunoreactivity was detected in cells of the medial hypothalamus with many of the same cells also displaying GLUT2 immunoreactivity. Together, these studies provide evidence for upstream GK promoter activity, GK mRNA, and GK itself in certain neuroendocrine cells outside the pancreatic islet and lead us to suggest that GK may play a broader role in glucose sensing by neuroendocrine cells than was thought previously.

Animals↗