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      High Glucose Induces Type 1 Hexokinase Gene Expression in Isolated Glomeruli of Diabetic Rats and in Mesangial Cells

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          Abstract

          Background: Diabetic nephropathy, which is characterized by renal hypertrophy and accumulation of extracellular matrix, is one of the leading causes for end-stage renal disease. Pathophysiological changes, which finally lead to the development of diabetic nephropathy, act through an increase in the intracellular NADH/NAD ratio and the activation of the polyol and protein kinase C pathways. The first rate-limiting enzymes in intracellular glucose metabolism are the hexokinases, which catalyze the phosphorylation of glucose. Therefore, in order to examine a possible link between increased glucose metabolism and the development of diabetic nephropathy mRNA and protein expression as well as enzyme activity of type 1 hexokinase were examined in kidneys of control and diabetic rats and in mesangial cells. Methods: Diabetes in rats was induced by intravenous injection of streptozotocin and animals were treated or not treated with insulin. RNA or protein was extracted from isolated glomeruli at different time intervals. In addition, glomerular mesangial cells were incubated in high glucose medium and hexokinase expression determined along with enzyme activity. Results: The experiments demonstrate a significant increase in gene and protein expression of type 1 hexokinase in glomeruli of diabetic rats throughout a three week observation period. Insulin therapy reduced glomerular type 1 hexokinase mRNA expression. Gene expression and hexokinase enzyme activity were also increased in mesangial cells grown in high glucose medium. Conclusion: The present experiments demonstrate that the expression of type 1 hexokinase is increased in isolated glomeruli of diabetic animals and is regulated by high ambient glucose concentrations. These results add further evidence to the fact that the kidney is one of the tissues most sensitive to high glucose levels in diabetes.

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          Most cited references 8

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          Molecular mechanisms of tumor necrosis factor alpha gene expression in monocytic cells via hyperglycemia-induced oxidant stress-dependent and -independent pathways.

           W Bai,  M Guha,  J. Nadler (2000)
          Increased oxidative stress has been reported in vivo in the diabetic state via the production of reactive oxygen species (ROS). Such stress is bound to play a key role on activation of circulating monocytes, leading to the accelerated atherosclerosis observed in diabetics. However the exact molecular mechanisms of monocyte activation by high glucose is currently unclear. Here, we demonstrate that chronic high glucose (CHG) causes a dramatic increase in the release of the inflammatory cytokine tumor necrosis factor alpha (TNFalpha), at least in part through enhanced TNFalpha mRNA transcription, mediated by ROS via activation of transcription factors nuclear factor kappaB (NF-kappaB) and activating protein-1 (AP-1). TNFalpha accumulation in the conditioned media was increased 10-fold and mRNA levels were increased 11.5-fold by CHG. The following observations supported that both NF-kappaB and AP-1 mediated enhanced TNFalpha transcription by CHG: 1) A 295-base pair fragment of the proximal TNFalpha promoter containing NF-kappaB and AP-1 sites reproduced the effects of CHG on TNFalpha transcription in a luciferase reporter assay, 2) mutational analyses of both NF-kappaB and the AP-1 sites abrogated 90% of the luciferase activity, 3) gel-shift analysis using the binding sites showed activation of NF-kappaB and AP-1 in CHG nuclear extracts, and 4) Western blot analyses demonstrated elevated nuclear levels of p65 and p50 and decreased cytosolic levels of IkappaBalpha in CHG-treated monocytes. That ROS acted as a key intermediate in the CHG pathway was supported by the following evidence: 1) increased superoxide levels similar to those observed with PMA or TNFalpha, 2) increased phosphorylation of stress-responsive mitogen-activated protein kinases p38 and JNK-1, 3) counteraction of the effects of CHG on TNFalpha production, the 295TNFluc reporter activity, activation of NF-kappaB, and repression of IkappaBalpha by antioxidants and p38 mitogen-activated protein kinase inhibitors. The study suggests that ROS function as key components in the regulatory pathway progressing from elevated glucose to monocyte activation.
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            Renal glucose production and utilization: new aspects in humans.

            According to current textbook wisdom the liver is the exclusive site of glucose production in humans in the postabsorptive state. Although many animal and in vitro data have documented that the kidney is capable of gluconeogenesis, production of glucose by the human kidney in the postabsorptive state has generally been regarded as negligible. This traditional view is based on net balance measurements which, other than after a prolonged fast or during metabolic acidosis, showed no significant net renal glucose release. However, recent studies have refuted this view by combining isotopic and balance techniques, which have demonstrated that renal glucose production accounts for 25% of systemic glucose production. Moreover, these studies indicate that glucose production by the human kidney is stimulated by epinephrine, inhibited by insulin and is excessive in diabetes mellitus. Since renal glucose release is largely, if not exclusively, due to gluconeogenesis, it is likely that the kidney is as important a gluconeogenic organ as the liver. The most important renal gluconeogenic precursors appear to be lactate, glutamine and glycerol. The implications of these recent findings on the understanding of the physiology and pathophysiology of human glucose metabolism are discussed.
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              Regulation of hexokinase I: crystal structure of recombinant human brain hexokinase complexed with glucose and phosphate.

               C Zeng,  G Fromm,  H Bartunik (1998)
              Hexokinase I, the pacemaker of glycolysis in brain tissue and red blood cells, is comprised of two similar domains fused into a single polypeptide chain. The C-terminal half of hexokinase I is catalytically active, whereas the N-terminal half is necessary for the relief of product inhibition by phosphate. A crystalline complex of recombinant human hexokinase I with glucose and phosphate (2.8 A resolution) reveals a single binding site for phosphate and glucose at the N-terminal half of the enzyme. Glucose and phosphate stabilize the N-terminal half in a closed conformation. Unexpectedly, glucose binds weakly to the C-terminal half of the enzyme and does not by itself stabilize a closed conformation. Evidently a stable, closed C-terminal half requires either ATP or glucose 6-phosphate along with glucose. The crystal structure here, in conjunction with other studies in crystallography and directed mutation, puts the phosphate regulatory site at the N-terminal half, the site of potent product inhibition at the C-terminal half, and a secondary site for the weak interaction of glucose 6-phosphate at the N-terminal half of the enzyme. The relevance of crystal structures of hexokinase I to the properties of monomeric hexokinase I and oligomers of hexokinase I bound to the surface of mitochondria is discussed. Copyright 1998 Academic Press.
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                Author and article information

                Journal
                NEP
                Nephron Physiol
                10.1159/issn.1660-2137
                Nephron Physiology
                S. Karger AG
                1660-2137
                2003
                March 2003
                31 March 2003
                : 93
                : 3
                : p67-p75
                Affiliations
                Department of Internal Medicine, Division of Nephrology and Osteology, University Hospital, Hamburg, Germany
                Article
                69555 Nephron Physiol 2003;93:p67–p75
                10.1159/000069555
                12660493
                © 2003 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 7, Tables: 1, References: 34, Pages: 1
                Product
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/69555
                Categories
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