High Glucose Stimulates Angiotensinogen Gene Expression and Cell Hypertrophy via Activation of the Hexosamine Biosynthesis Pathway in Rat Kidney Proximal Tubular Cells

The present study investigated whether activation of the hexosamine biosynthesis pathway might mediate at least in part the high glucose effect on angiotensinogen (ANG) gene expression and immortalized renal proximal tubular cell (IRPTC) hypertrophy. IRPTC were cultured in monolayer. ANG, renin, and β-actin mRNA expression were determined by specific RT-PCR assays. Phosphorylation of p38 MAPK, activating transcription factor-2 (ATF-2), and cAMP-responsive element-binding protein (CREB) was determined by Western blot analysis. Cell hypertrophy was assessed by flow cytometry, intracellular p27kip1 protein levels, and [3H]leucine incorporation into proteins. Glucosamine stimulated ANG and renin mRNA expression and enhanced p38 MAPK, ATF-2, and CREB phosphorylation in normal glucose (5 mm) medium. Azaserine and 6-diazo-5-oxo-l-norleucine (inhibitors of glutamine: fructose-6-phosphate amino transferase enzyme) blocked the stimulatory effect of high glucose, but not that of glucosamine, on ANG gene expression in IRPTCs. SB 203580 (a specific p38 MAPK inhibitor) attenuated glucosamine action on ANG gene expression as well as p38 MAPK and ATF-2 phosphorylation, but not that of CREB. GF 109203X and calphostin C (inhibitors of protein kinase C) blocked the effect of glucosamine on ANG gene expression and CREB phosphorylation, but had no impact on p38 MAPK and ATF-2 phosphorylation. Finally, both glucosamine and high glucose induced IRPTC hypertrophy. The hypertrophic effect of glucosamine was blocked in the presence of GF 109203X, but not azaserine and SB 203580. In contrast, the hypertrophic effect of high glucose was blocked in the presence of azaserine and GF 109203X, but not SB203580. Our studies demonstrate that the stimulatory effect of high glucose on ANG gene expression and IRPTC hypertrophy may be mediated at least in part via activation of hexosamine biosynthesis pathway signaling.

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Glucose regulates expression of pro-inflammatory genes IL-1β and IL-12 through a mechanism involving hexosamine biosynthesis pathway dependent regulation of α-E catenin in the RAW 264.7 macrophage cell line

10.1101/2021.04.14.439728 ◽

2021 ◽

Author(s):

Waruni C. Dissanayake ◽

Jin Kyo Oh ◽

Brie Sorrenson ◽

Peter R. Shepherd

Keyword(s):

Gene Expression ◽

High Glucose ◽

Inflammatory Responses ◽

Macrophage Polarization ◽

Mrna Level ◽

Biosynthesis Pathway ◽

Glucose Levels ◽

Raw264.7 Macrophages ◽

Hexosamine Biosynthesis ◽

Hexosamine Biosynthesis Pathway

AbstractHigh glucose levels are associated with changes in macrophage polarization and evidence indicates that the sustained or even short-term high glucose levels modulate inflammatory responses in macrophages. However, the mechanism by which macrophages can sense the changes in glucose levels are not clearly understood. We find that high glucose levels rapidly increase the α-E catenin protein level in RAW264.7 macrophages. We also find an attenuation of glucose induced increase of α-E catenin when hexosamine biosynthesis pathway is inhibited either with glutamine depletion or with the drugs azaserine and tunicamycin. This indicates the involvement of hexosamine biosynthesis pathway in this process. Then, we investigated the potential role of α-E catenin in glucose induced macrophage polarization. We find that the reduction of α-E catenin level using siRNA attenuates the glucose induced change of IL-1β mRNA level under LPS stimulated condition. Further, we identified that the depletion of α-E catenin also decreases the IL-12 gene expression in basal glucose conditions leading to a reduction of glucose induced changes in IL-12. Together this indicates that α-E catenin can sense the changes in glucose levels in macrophages via hexosamine biosynthesis pathway and also can modulate the glucose induced gene expression of inflammatory markers such as IL-1-β and IL-12. This identifies a new part of the mechanism by which macrophages are able to respond to changes in glucose levels.

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High Glucose Stimulates Angiotensinogen Gene Expression via Reactive Oxygen Species Generation in Rat Kidney Proximal Tubular Cells

Endocrinology ◽

10.1210/endo.143.8.8931 ◽

2002 ◽

Vol 143 (8) ◽

pp. 2975-2985 ◽

Cited By ~ 90

Author(s):

Tusty-Jiuan Hsieh ◽

Shao-Ling Zhang ◽

Janos G. Filep ◽

Shiow-Shih Tang ◽

Julie R. Ingelfinger ◽

...

Keyword(s):

Gene Expression ◽

P38 Mapk ◽

High Glucose ◽

Ros Generation ◽

Proximal Tubular Cells ◽

Mapk Phosphorylation ◽

Tubular Cells ◽

Proximal Tubular ◽

Kidney Proximal Tubular Cells ◽

Sb 203580

Abstract The present studies investigated whether the effect of high glucose levels on angiotensinogen (ANG) gene expression in kidney proximal tubular cells is mediated via reactive oxygen species (ROS) generation and p38 MAPK activation. Rat immortalized renal proximal tubular cells (IRPTCs) were cultured in monolayer. Cellular ROS generation and p38 MAPK phosphorylation were assessed by lucigenin assay and Western blot analysis, respectively. The levels of immunoreactive rat ANG secreted into the media and cellular ANG mRNA were determined by a specific RIA and RT-PCR, respectively. High glucose (25 mm) evoked ROS generation and p38 MAPK phosphorylation as well as stimulated immunoreactive rat ANG secretion and ANG mRNA expression in IRPTCs. These effects of high glucose were blocked by antioxidants (taurine and tiron), inhibitors of mitochondrial electron transport chain complex I (rotenone) and II (thenoyltrifluoroacetone), an inhibitor of glycolysis-derived pyruvate transport into mitochondria (α-cyano-4-hydroxycinnamic acid), an uncoupler of oxidative phosphorylation (carbonyl cyanide m-chlorophenylhydrazone), a manganese superoxide dismutase mimetic, catalase, and a specific inhibitor of p38 MAPK (SB 203580), but were not affected by an inhibitor of the malate-aspartate shuttle (aminooxyacetate acid). Hydrogen peroxide (≥10−5m) also stimulated p38 MAPK phosphorylation, ANG secretion, and ANG mRNA gene expression, but its stimulatory effect was blocked by catalase and SB 203580. These studies demonstrate that the stimulatory action of high glucose on ANG gene expression in IRPTCs is mediated at least in part via ROS generation and subsequent p38 MAPK activation.

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Glucose regulates expression of pro-inflammatory genes IL-1β and IL-12 through a mechanism involving hexosamine biosynthesis pathway dependent regulation of αEcatenin

Bioscience Reports ◽

10.1042/bsr20211066 ◽

2021 ◽

Author(s):

Waruni C Dissanayake ◽

Jin Kyo Oh ◽

Brie Sorrenson ◽

Peter R Shepherd

Keyword(s):

High Glucose ◽

Inflammatory Responses ◽

Macrophage Polarization ◽

Mrna Levels ◽

Biosynthesis Pathway ◽

Glucose Levels ◽

Raw264.7 Macrophages ◽

Hexosamine Biosynthesis ◽

Induced Changes ◽

Hexosamine Biosynthesis Pathway

High glucose levels are associated with changes in macrophage polarization and evidence indicates that the sustained or even short-term high glucose levels modulate inflammatory responses in macrophages. However, the mechanism by which macrophages can sense the changes in glucose levels are not clearly understood. We find that high glucose levels rapidly increase the α-E catenin protein level in RAW264.7 macrophages. We also find an attenuation of glucose induced increase of α-E catenin when hexosamine biosynthesis pathway is inhibited either with glutamine depletion or with the drugs azaserine and tunicamycin. This indicates the involvement of hexosamine biosynthesis pathway in this process. Then, we investigated the potential role of α-E catenin in glucose induced macrophage polarization. We find that the reduction of α-E catenin level using siRNA attenuates the glucose induced changes of both IL-1β and IL-12 mRNA levels under LPS stimulated condition but does not affect TNF-α expression. Together this indicates that α-E catenin can sense the changes in glucose levels in macrophages via hexosamine biosynthesis pathway and also can modulate the glucose induced gene expression of inflammatory markers such as IL-1β and IL-12. This identifies a new part of the mechanism by which macrophages are able to respond to changes in glucose levels.

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High glucose and insulin promote O-GlcNAc modification of proteins, including α-tubulin

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00382.2002 ◽

2003 ◽

Vol 284 (2) ◽

pp. E424-E434 ◽

Cited By ~ 100

Author(s):

Jennie L. E. Walgren ◽

Timothy S. Vincent ◽

Kevin L. Schey ◽

Maria G. Buse

Keyword(s):

Posttranslational Modifications ◽

High Glucose ◽

Biosynthesis Pathway ◽

Two Dimensional Gel Electrophoresis ◽

Novel Proteins ◽

L6 Myotubes ◽

Hsp70 Family ◽

Glucose Flux ◽

Hexosamine Biosynthesis ◽

Hexosamine Biosynthesis Pathway

Increased flux through the hexosamine biosynthesis pathway has been implicated in the development of glucose-induced insulin resistance and may promote the modification of certain proteins with O-linked N-acetylglucosamine ( O-GlcNAc). L6 myotubes (a model of skeletal muscle) were incubated for 18 h in 5 or 25 mM glucose with or without 10 nM insulin. As assessed by immunoblotting with an O-GlcNAc-specific antibody, high glucose and/or insulin enhanced O-GlcNAcylation of numerous proteins, including the transcription factor Sp1, a known substrate for this modification. To identify novel proteins that may be O-GlcNAc modified in a glucose concentration/insulin-responsive manner, total cell membranes were separated by one- or two-dimensional gel electrophoresis. Selected O-GlcNAcylated proteins were identified by mass spectrometry (MS) analysis. MS sequencing of tryptic peptides identified member(s) of the heat shock protein 70 (HSP70) family and rat α-tubulin. Immunoprecipitation/immunoblot studies demonstrated several HSP70 isoforms and/or posttranslational modifications, some with selectively enhanced O-GlcNAcylation following exposure to high glucose plus insulin. In conclusion, in L6 myotubes, Sp1, membrane-associated HSP70, and α-tubulin are O-GlcNAcylated; the modification is markedly enhanced by sustained increased glucose flux.

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Increased flux through the hexosamine biosynthesis pathway inhibits glucose transport acutely by activation of protein kinase C

Biochemical Journal ◽

10.1042/bj3240981 ◽

1997 ◽

Vol 324 (3) ◽

pp. 981-985 ◽

Cited By ~ 41

Author(s):

Anthony FILIPPIS ◽

Stella CLARK ◽

Joseph PROIETTO

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Protein Kinase ◽

Glucose Transport ◽

High Glucose ◽

Biosynthesis Pathway ◽

Rat Adipocytes ◽

Kinase C ◽

Hexosamine Biosynthesis ◽

Hexosamine Biosynthesis Pathway

The hexosamine biosynthesis pathway and protein kinase C (PKC) activation mediate hyperglycaemia-induced impaired glucose transport, but the relative role of each pathway is unknown. Following a 2 h preincubation of rat adipocytes in the presence of either high glucose (30 mM) plus insulin (0.7 nM) or glucosamine (3 mM), both high glucose and glucosamine inhibited subsequent basal and insulin-stimulated glucose transport, measured at 5.0 mM glucose. Azaserine, an inhibitor of the enzyme glutamine:fructose-6-phosphate aminotransferase, abolished the effect of high glucose, but not that of glucosamine. Ro-31-8220, an inhibitor of PKC, reversed the effects of both high glucose and glucosamine, suggesting that flux through the hexosamine biosynthesis pathway impaired glucose transport acutely by activating PKC. Both high glucose and glucosamine caused a 3-fold increase in PKC activity; this effect of high glucose, but not that of glucosamine, was partially decreased by azaserine. Neither high glucose nor glucosamine altered basal or insulin-stimulated plasma membrane GLUT1 levels, whereas both treatments decreased basal, but not insulin-stimulated, GLUT4 levels. Azaserine abolished the effect of high glucose, but not that of glucosamine, on basal plasma membrane GLUT4 levels. Ro-31-8220, which returned glucose transport to control values, caused a further decrease in plasma membrane GLUT4 levels. It is concluded that, in rat adipocytes, an acute increase in flux through the hexosamine biosynthesis pathway inhibits glucose transport by activation of PKC.

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