The effect of modulators of inflammation on hepatic acute phase proteins and metabolic enzymes
Date
2010-03
Authors
Visser, Jacobus Albertus Koch
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : University of Stellenbosch
Abstract
ENGLISH ABSTRACT:
Crosstalk exists between the stress- and immune-system and this crosstalk has pharmacological
importance in the use of glucocorticoids (GCs) as anti-inflammatory drugs for diseases such as
asthma and arthritis. The focus of studies on this crosstalk has mainly been on the effects of GCs
on immune function. The effect of the immune system on GC action, especially in the periphery, is
not as well studied. The liver plays an important role in inflammation and stress in producing the
acute phase proteins (APPs) required for the resolution of inflammation as well as in producing
systemic glucose, through gluconeogenesis, required to fuel the stress responses. Understanding
effects of stress and inflammation and their interplay in the liver is thus not only useful to expand
our understanding of these systems but could also have clinical applications in understanding the
side-effects associated with pharmacological use of GCs. CpdA has been identified as a selective
glucocorticoid receptor (GR) modulator (SEGRM) in that it is able to repress genes but is not
capable of activating genes via the GR. This attribute suggests that CpdA has the potential to be
developed as an anti-inflammatory drug that displays fewer side effects. The current study
investigated and compared effects of dexamethasone, a potent GR agonist, and CpdA, in the
presence and absence of interleukin 6 (IL6), on the glucocorticoid receptor, three metabolic enzyme
genes, involved in gluconeogenesis, and three APP genes. The metabolic enzyme genes
investigated were tyrosine amintotransferase (TAT), phosphoenolpyruvate carboxykinase (PEPCK),
and gamma glutmayltransferase (GGT), while the APP genes were serum amyloid A (SAA), Creactive
protein (CRP), and corticosteroid-binding globulin (CBG). The study investigated effects
at the protein level, using Western blotting and ELISA assays, the protein activity level, using
enzyme activity assays and whole cell binding, and at the mRNA level, using quantitive polymerase
chain reactions (qPCR), in a mouse hepatoma cell line (BWTG3). The study showed that
dexamethasone (Dex) and IL6 generally have divergent effects on the GR and metabolic enzymes Crosstalk exists between the stress- and immune-system and this crosstalk has pharmacological
importance in the use of glucocorticoids (GCs) as anti-inflammatory drugs for diseases such as
asthma and arthritis. The focus of studies on this crosstalk has mainly been on the effects of GCs
on immune function. The effect of the immune system on GC action, especially in the periphery, is
not as well studied. The liver plays an important role in inflammation and stress in producing the
acute phase proteins (APPs) required for the resolution of inflammation as well as in producing
systemic glucose, through gluconeogenesis, required to fuel the stress responses. Understanding
effects of stress and inflammation and their interplay in the liver is thus not only useful to expand
our understanding of these systems but could also have clinical applications in understanding the
side-effects associated with pharmacological use of GCs. CpdA has been identified as a selective
glucocorticoid receptor (GR) modulator (SEGRM) in that it is able to repress genes but is not
capable of activating genes via the GR. This attribute suggests that CpdA has the potential to be
developed as an anti-inflammatory drug that displays fewer side effects. The current study
investigated and compared effects of dexamethasone, a potent GR agonist, and CpdA, in the
presence and absence of interleukin 6 (IL6), on the glucocorticoid receptor, three metabolic enzyme
genes, involved in gluconeogenesis, and three APP genes. The metabolic enzyme genes
investigated were tyrosine amintotransferase (TAT), phosphoenolpyruvate carboxykinase (PEPCK),
and gamma glutmayltransferase (GGT), while the APP genes were serum amyloid A (SAA), Creactive
protein (CRP), and corticosteroid-binding globulin (CBG). The study investigated effects
at the protein level, using Western blotting and ELISA assays, the protein activity level, using
enzyme activity assays and whole cell binding, and at the mRNA level, using quantitive polymerase
chain reactions (qPCR), in a mouse hepatoma cell line (BWTG3). The study showed that
dexamethasone (Dex) and IL6 generally have divergent effects on the GR and metabolic enzymes
AFRIKAANSE OPSOMMING: Kruiskommunikasie bestaan tussen die stres– en die immuunsisteem en hierdie kruiskommunikasie is van farmakologiese belang vir die gebruik van glukokortikoïede (GKe) as anti-inflammatoriese medikasie vir siektes soos asma en artritis. Tot dusver was die fokus van studies oor hierdie kruiskommunikasie hoofsaaklik op die effek van GKe op immuunfunksie. Die effek van die immuunsisteem op GK werking, veral in die periferie, is nie so goed bestudeer nie. Die lewer speel ʼn belangrike rol in inflammasie en stres deurdat dit die akute fase proteïene (AFPs) produseer wat benodig word vir die resolusie van inflammasie en omdat dit ook sistemiese glukose produseer, d.m.v. glukoneogenese, wat benodig word om die stres reaksie te dryf. ’n Beter insig in die effek van stres en inflammasie sowel as hul interaksie in die lewer is dus handig, nie net om ons begrip van hierdie sisteme te verbeter nie, maar ook omdat dit kliniese toepassing kan hê deurdat dit ons begrip van die newe-effekte wat gepaard gaan met die farmakologiese gebruik van GKe verbeter. Verbinding A (CpdA) is geïdentifiseer as ʼn selektiewe glukokortikoïed reseptor (GR) moderator (SERGM) omdat dit die vermoë het om gene te onderdruk maar nie te aktiveer d.m.v. die GR. Hierdie eienskap dui op die potensiaal van CpdA om ontwikkel te word as ʼn anti-inflammatoriese middel met minder newe-effekte. Die huidige studie het die effekte van dexamethasone, ʼn sterk GR agonis, en CpdA, beide in die teenwoordigheid en afwesigheid van interleukin 6 (IL6), op die GR, drie metaboliese ensiem gene wat betrokke is by glukoneogenese, sowel as drie APP gene, ondersoek en vergelyk. Die metaboliese ensiem gene wat ondersoek is, is tirosien aminotransferase (TAT), fosfoenolpirovaat karboksikinase (PEPCK), en gamma glutamieltransferase (GGT), terwyl die APP gene serum amiloïede A (SAA), C-reaktiewe proteïen (CRP), en kortikosteroïed bindings globien (CBG) was. Die studie het die effekte in ʼn muis hepatoma sellyn (BWTG3) op die proteïen vlak, deur van Western blotting en ELISA essays gebruik te maak, die proteïen aktiwiteits vlak, deur van ensiem aktiwiteits essays en vol-sel binding gebruik te maak, sowel as op die mRNA vlak, deur van kwantitatiewe polimerase ketting reaksie (qPCR) gebruik te maak, ondersoek. Die studie toon dat dexamethasone (Dex) en IL6 in die algemeen divergente effekte het op die GR en metaboliese ensieme deurdat Dex GR af-reguleer en die metaboliese ensieme op-reguleer, terwyl IL6 die GR op-reguleer en die metaboliese ensieme af-reguleer, en dat hulle funksies konvergerend is vir die APPs deurdat beide positiewe APPs opreguleer en negatiewe APPs afreguleer. In teenstelling met Dex het CpdA die GR op-gereguleer en die metaboliese ensieme af-gereguleer terwyl dit, soos Dex, die positiewe APPs op-gereguleer en die negatiewe APPs af-gereguleer het. Ons resultate vir Dex en IL6 word ondersteun deur vorige werk in die literatuur. Ons studie is wel uniek omdat dit die ondersoek van drie metaboliese ensieme kombineer met die ondersoek van drie APPs, sowel as GR vlakke in ʼn enkele sisteem onder dieselfde eksperimentele kondisies. Verder het ons resultate met CpdA verskeie nuwe aspekte, soos die af-regulering van metaboliese gene, opgelewer wat bydra tot die groeiende poel van kennis oor hierdie ongewone GR ligand en die moontlike farmakologiese gebruik daarvan.
AFRIKAANSE OPSOMMING: Kruiskommunikasie bestaan tussen die stres– en die immuunsisteem en hierdie kruiskommunikasie is van farmakologiese belang vir die gebruik van glukokortikoïede (GKe) as anti-inflammatoriese medikasie vir siektes soos asma en artritis. Tot dusver was die fokus van studies oor hierdie kruiskommunikasie hoofsaaklik op die effek van GKe op immuunfunksie. Die effek van die immuunsisteem op GK werking, veral in die periferie, is nie so goed bestudeer nie. Die lewer speel ʼn belangrike rol in inflammasie en stres deurdat dit die akute fase proteïene (AFPs) produseer wat benodig word vir die resolusie van inflammasie en omdat dit ook sistemiese glukose produseer, d.m.v. glukoneogenese, wat benodig word om die stres reaksie te dryf. ’n Beter insig in die effek van stres en inflammasie sowel as hul interaksie in die lewer is dus handig, nie net om ons begrip van hierdie sisteme te verbeter nie, maar ook omdat dit kliniese toepassing kan hê deurdat dit ons begrip van die newe-effekte wat gepaard gaan met die farmakologiese gebruik van GKe verbeter. Verbinding A (CpdA) is geïdentifiseer as ʼn selektiewe glukokortikoïed reseptor (GR) moderator (SERGM) omdat dit die vermoë het om gene te onderdruk maar nie te aktiveer d.m.v. die GR. Hierdie eienskap dui op die potensiaal van CpdA om ontwikkel te word as ʼn anti-inflammatoriese middel met minder newe-effekte. Die huidige studie het die effekte van dexamethasone, ʼn sterk GR agonis, en CpdA, beide in die teenwoordigheid en afwesigheid van interleukin 6 (IL6), op die GR, drie metaboliese ensiem gene wat betrokke is by glukoneogenese, sowel as drie APP gene, ondersoek en vergelyk. Die metaboliese ensiem gene wat ondersoek is, is tirosien aminotransferase (TAT), fosfoenolpirovaat karboksikinase (PEPCK), en gamma glutamieltransferase (GGT), terwyl die APP gene serum amiloïede A (SAA), C-reaktiewe proteïen (CRP), en kortikosteroïed bindings globien (CBG) was. Die studie het die effekte in ʼn muis hepatoma sellyn (BWTG3) op die proteïen vlak, deur van Western blotting en ELISA essays gebruik te maak, die proteïen aktiwiteits vlak, deur van ensiem aktiwiteits essays en vol-sel binding gebruik te maak, sowel as op die mRNA vlak, deur van kwantitatiewe polimerase ketting reaksie (qPCR) gebruik te maak, ondersoek. Die studie toon dat dexamethasone (Dex) en IL6 in die algemeen divergente effekte het op die GR en metaboliese ensieme deurdat Dex GR af-reguleer en die metaboliese ensieme op-reguleer, terwyl IL6 die GR op-reguleer en die metaboliese ensieme af-reguleer, en dat hulle funksies konvergerend is vir die APPs deurdat beide positiewe APPs opreguleer en negatiewe APPs afreguleer. In teenstelling met Dex het CpdA die GR op-gereguleer en die metaboliese ensieme af-gereguleer terwyl dit, soos Dex, die positiewe APPs op-gereguleer en die negatiewe APPs af-gereguleer het. Ons resultate vir Dex en IL6 word ondersteun deur vorige werk in die literatuur. Ons studie is wel uniek omdat dit die ondersoek van drie metaboliese ensieme kombineer met die ondersoek van drie APPs, sowel as GR vlakke in ʼn enkele sisteem onder dieselfde eksperimentele kondisies. Verder het ons resultate met CpdA verskeie nuwe aspekte, soos die af-regulering van metaboliese gene, opgelewer wat bydra tot die groeiende poel van kennis oor hierdie ongewone GR ligand en die moontlike farmakologiese gebruik daarvan.
Description
Thesis (MSc (Biochemistry))--University of Stellenbosch, 2010.
Keywords
Inflammation, Stress, Glucocorticoids, Compound A, Dissertations -- Biochemistry, Theses -- Biochemistry