Thesis (MSc)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: Introduction: Exercise increases skeletal muscle glucose uptake via AMP-activated
protein kinase (AMPK) activation and GLUT4 translocation from cytosol to cell
membrane. It also promotes glucose utilisation in type 2 diabetic patients via
increased insulin sensitivity. Insulin stimulates GLUT4 translocation by activating P13-
kinase and protein kinase B (PKB/Akt). We therefore postulated that a connection
exists between these two pathways upstream of GLUT4 translocation. Understanding
this connection is important in the development of treatment strategies for type 2
diabetes. This exercise-induced increase in AMP-activated protein kinase (AMPK)
activation can be mimicked by a pharmacological agent, 5'-aminoimidazole-4-
carboxamide ribonucleoside (AlGAR), which is converted intracellularly into 5'-
aminoimidazole-4-carboxamide-ribonucleosidephosphate (ZMP), an AMP analogue.
Aim: To investigate the effect of two pharmacological AMPK-activating compounds,
ZMP and AlGAR, on the phosphorylation of AMPK, the phosphorylation of PKB/Akt
as well as possible feedback on insulin-stimulated glucose uptake and GLUT4
translocation.
Materials and Methods: Adult ventricular cardiomyocytes were isolated from male
Wistar rats by collagenase perfusion and treated with 1 mM AlGAR or 1 mM ZMP in
the presence or absence of 100 nM insulin or 100 nM wortmannin, an inhibitor of P13-
kinase. Glucose uptake was measured via eH]-2-deoxyglucose (2DG) accumulation.
PKB/Akt and AMPK phosphorylation and GLUT4 translocation was detected by
Western blotting. Purinergic receptors were blocked with 8-cyclopentyl-1,3- dipropylxanthine (8CPT) and the effect on AMPK phosphorylation noted. Certain
results were confinned or refuted by repeating experiments using the isolated rat
heart model.
Results: AICAR and ZMP promoted AMPK phosphorylation. Neither drug increased
glucose uptake but in fact inhibited basal glucose uptake, although GLUT4
translocation from cytosol to membrane occurred. Both compounds also attenuated
insulin stimulated glucose uptake. Wortmann in abolished glucose uptake and
PKB/Akt phosphorylation elicited by insulin while, in the presence of wortmannin,
AICAR and ZMP increased levels of PKB/Akt phosphorylation. Although AICAR and
ZMP increased glucose uptake in skeletal muscle, this was not seen in
cardiomyocytes. However both compounds increased GLUT4 translocation, clearly
demonstrating that translocation and activation of GLUT4 are separate processes.
8CPT had no effect on the phosphorylation of AMPK by either AICAR or ZMP
indicating that there was no involvement of the purinergic receptors.
Conclusion: Although AICAR and ZMP increase glucose uptake in skeletal muscle,
this was not seen in cardiomyocytes. Conversely, both compounds inhibited both
basal and insulin stimulated glucose uptake despite increasing GLUT4 translocation.
Inhibition of PI3-kinase in presence or absence of insulin unmasked hitherto
unknown effects of AICAR and ZMP on PKB phosphorylation. / AFRIKAANSE OPSOMMING:
Agtergrond:
Oefening verhoog skeletspier glukose opname via AMP-geaktiveerde
protein kinase (AMPK) aktivering en GLUT4 translokering vanaf die sitosol na die
selmembraan. Dit verbeter ook glukose verbruik in tipe 2 diabetes pasiënte via
verhoogde insulien sensitiwiteit. Insulien stimuleer GLUT4 translokering deur P13-
kinase en protein kinase B (PKB/Akt) te aktiveer. Dit word dus gepostuleer dat daar
'n verbinding tussen hierdie twee paaie, wat beide betrokke is by GLUT4
translokering, bestaan. Dit is belangrik om hierdie verbinding te verstaan aangesien
dit in behandelingstrategieë van tipe 2 diabetes geteiken kan word. Die oefening
geïnduseerde verhoging in AMPK aktivering, kan deur 'n farmakologiese middel 5'-
aminoimidasool-4-karboksamied ribonukleosied (AICAR), wat intrasellulêr omgesit
word na 5'-aminoimidasool-4-karboksamied-ribonukleosiedfosfaat (ZMP), 'n AMP
analoog, nageboots word.
Doel:
Om die effek van twee farmakologiese AMPK-aktiveringsmiddels, AICAR en
ZMP, op die fosforilering van AMPK en PKB/Akt, sowel as moontlike effekte daarvan
op insulien-gestimuleerde glukose opname en GLUT4 translokering, te ondersoek.
Materiale en Metodes:
Volwasse ventrikulêre kardiomiosiete is uit manlike Wistar
rotharte geïsoleer d.m.v kollagenase perfusies en behandel met 1 mM AICAR of 1
mM ZMP in die teenwoordigheid of afwesigheid van 100 nM insulien of 100 nM
wortmannin. Glukose opname is gemeet via intrasellulêre [3H]-2-deoksiglukose
akkumulasie; PKB/Akt en AMPK fosforilering sowel as GLUT4 translokering is bepaal
deur Western blot analises. Purinergiese reseptore is geblokkeer met 8-siklopentiel-
1,3-dipropielxanthien (8CPT) en die effek daarvan op AMPK fosforilering genoteer. Ten einde resultate wat in die geïsoleerde kardiomiosiet-model verkry is, te bevestig,
is sekere eksperimente in die geïsoleerde rothart herhaal.
Resultate:
Beide AIGAR en ZMP stimuleer AMPK fosforilering. Die middels kan nie
glukose opname verhoog nie, inteendeel, basale glukose opname is onderdruk
alhoewel GLUT4 translokering vanaf die sitosol na die selmembraan wel plaasgevind
het. Wortmannin kon insulien gemedieerde glukose opname en PKB/Akt fosforilering
onderdruk. In die teenwoordigheid van wortmannin het beide AIGAR en ZMP
PKB/Akt fosforilering verhoog. Alhoewel beide AIGAR en ZMP glukose opname in
skeletspier verhoog, was dit nie die geval in kardiomiosiete nie. Beide middels het
wel GLUT 4 translokering verhoog, wat duidelik demonstreer dat die translokering en
aktivering van GLUT4, verskillende prosesse is. 8GPT het geen effek gehad op die
fosforilering van AMPK deur AIGAR of ZMP nie, wat bewys dat daar geen
betrokkenheid van die purinergiese reseptore was nie.
Gevolgtrekking:
Alhoewel AIGAR en ZMP glukose opname in skeletspier verhoog is
dit nie die geval in kardiomiosiete nie. Beide middels inhibeer basale en insuliengestimuleerde
glukose opname maar stimuleer GLUT4 translokeering. Inhibisie van
PI3-kinase in die teenwoordigheid of afwesigheid van insulien, ontmasker voorheen
onbekende effekte van AIGAR en ZMP op PKB/Akt fosforilering.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/50483 |
| Date | 03 1900 |
| Creators | Webster, Ingrid |
| Contributors | Huisamen, Barbara, Lochner, Amanda, Stellenbosch University. Faculty of Medicine & Health Sciences. Dept. of Biomedical Sciences. Medical Physiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 168 p. : ill. |
| Rights | Stellenbosch University |
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