| Summary: | Thesis (MScMedSc)--Stellenbosch University, 2012. === ENGLISH ABSTRACT: Introduction: The worldwide escalation in the incidence of obesity and its strong association
with insulin resistance, type 2 diabetes and the cardiovascular complications that accompany
these disease states have elicited interest in the underlying mechanisms of these pathologies.
Preliminary data generated in our laboratory showed that obesity is associated with
abnormalities in the insulin signalling pathway. Specifically, we found a down-regulation of
protein kinase B (PKB/Akt), which is known to mediate the metabolic effects of insulin. One
of the downstream targets of PKB/Akt is glycogen synthase kinase-3 (GSK-3), which is
inhibited by this phosphorylation. Detrimental effects of unopposed activity of GSK-3 have
recently been described. This may play a pivotal role in some of the adverse consequences of
insulin resistance in the heart.
Hypothesis: Chronic inhibition of GSK-3 will induce myocardial hypertrophy or exacerbate
the development of existing hypertrophy in a pre-diabetic model of diet induced obesity and
insulin resistance.
Objectives: (1) Assess the extent of the development of myocardial hypertrophy in a rat
model of diet induced obesity (DIO) and insulin resistance. (2) Assess the effect of inhibition
of GSK-3 protein on the development of myocardial hypertrophy.
Methods: Two groups of age-matched male Wistar rats were used. Control animals received
standard rat chow, while obese animals received a high caloric diet for 20 weeks. After 12
weeks, half of the animals in both groups received GSK-3 inhibitor treatment (CHIR118637,
30mg/kg/day, Novartis). At the end of 20 weeks, three series of experiments were conducted.
(i) The animals were subjected to echocardiography to determine in vivo myocardial function,
and biometric, metabolic and biochemical parameters were evaluated. (ii) The ability of the cardiomyocytes to accumulate deoxy-glucose after stimulation with
insulin was determined, and (iii) the localization of key proteins was monitored using
fluorescence microscopy and cell size was determined using light microscopy and flow
activated cell sorter analysis.
Results and discussion: The high caloric diet increased body weight (p<0.005) and intraperitoneal
fat mass (p<0.01) when compared to controls. Complications associated with
obesity, such as impaired glucose tolerance (p<0.05), hyperinsulinemia (p<0.0005) and an
increased HOMA-IR index (p<0.01) were observed. Additionally, cardiomyocytes from the
DIO animals had a significantly impaired response to insulin, specifically when 10nM
(p<0.05) and 100nM (p<0.05) of insulin were used as stimulus. We also found a
dysregulation in PKB/Akt, indicated by a down-regulation of phosphorylated PKB/Akt
(p<0.01). The diet promoted the development of myocardial hypertrophy, since the
ventricular weight (p<0.05) and ventricular weight to tibia length ratio were increased
(p<0.01). Echocardiography experiments showed an increase in end diastolic diameter in the
DIO animals (p<0.05). Additionally, there was an increase in the cardiomyocyte cell width in
the DIO rats (p<0.0001) and a tendency for peri-nuclear localization of NFATc3. GSK-3
inhibition promoted the development of insulin resistance in control animals, as indicated by
an increase in the body weight (p<0.05), serum insulin levels (p<0.01) and HOMA-IR index
(p<0.01). In the DIO animals, the GSK-3 inhibitor treatment improved insulin resistance, as a
decrease in serum insulin concentration (p<0.05) was observed. The cardiomyocytes from the
treated DIO animals also showed an increase in glucose uptake (p<0.05) when stimulated
with 100nM of insulin. The GSK-3 inhibitor promoted the development of myocardial
hypertrophy in the control animals, indicated by an increase in ventricular weight (p<0.05)
and cardiomyocyte cell width (p<0.0001), but did not exacerbate hypertrophy in the DIO animals. Conclusion: Both the high caloric diet and the GSK-3 inhibitor promoted the development of
insulin resistance and myocardial hypertrophy in the rats. In the DIO animals the GSK-3
inhibitor treatment ameliorated insulin resistance and did not promote the further
development of myocardial hypertrophy. === AFRIKAANSE OPSOMMING: Inleiding: Die huidige styging in vetsugtigheid en die sterk assosiasie daarvan met insulien
weerstandigheid, tipe 2 diabetes en kardiovaskulêre komplikasies soos hipertrofie, het ‘n
belangstelling in die onderliggende meganismes van hierdie siektetoestande ontlok.
Voorlopige data uit ons laboratorium het getoon dat vetsug geassosieerd is met abnormaliteite
in die insulien seintransduksie-pad soos byvoorbeeld ‘n afregulering van miokardiale proteïen
kinase B (PKB/Akt), wat bekend is om die metaboliese effekte van insulien te medieer. Een
van die proteïene wat deur PKB/Akt gefosforileer en daardeur geïnhibeer word, is glikogeen
sintase kinase-3 (GSK-3). Negatiewe effekte van onge-opponeerde aktiwiteit van GSK-3 is
beskryf en dit mag ‘n sleutelrol speel in sommige van die nadelige gevolge van insulien
weerstandigheid in die hart.
Hipotese: Chroniese onderdrukking van GSK-3 sal miokardiale hipertrofie ontlok of die
bestaande hipertrofie in ‘n pre-diabetiese model van dieet-geïnduseerde vetsug en insulien
weerstandigheid vererger.
Doelstellings: (1) Om die omvang van die ontwikkeling van miokardiale hipertrofie in ‘n
rotmodel van dieet-geïnduseerde vetsug te ondersoek en (2) om die effek van inhibisie van
GSK-3 op die ontwikkeling van hipertrofie te ondersoek.
Metodes: Ouderdomsgepaarde manlike Wistarrotte is in hierdie studie gebruik. Die diere is
vir ‘n periode van 20 weke aan verskillende diëte onderwerp, naamlik standaard kommersiële
rotkos vir die kontrole diere en ‘n hoë kalorie dieet vir die eksperimenteel vet diere (DIO).
Helfte van elke groep diere is vir 8 weke met ‘n GSK-3 inhibitor behandel (CHIR118637,
30mg/kg/day, Novartis). Na die 20 weke is 3 eksperimentele reekse uitgevoer: (i) Die diere is
eggokardiografies ondersoek om in vivo miokardiale funksie te bepaal en biometriese,
metaboliese en biochemiese parameters is evalueer. (ii) Die vermoë van kardiomiosiete om de-oksiglukose na insulien stimulasie te akkumuleer,
is bepaal, en (iii) die lokalisering van sleutelproteïene is met behulp van fluoressensie
mikroskopie en die selgrootte met behulp van ligmikroskopie bepaal.
Resultate en bespreking: Die hoë kalorie dieet het gepaard gegaan met ‘n beduidende
toename in liggaamsgewig (p<0.005) en intraperitoneale vetmassa (p<0.01) in vergelyking
met diere op die kontrole dieet. Newe-effekte geassosieerd met vetsug nl. onderdrukte
glucose toleransie (p<0.05), hiperinsulinemie (p<0.0005) en ‘n verhoogde HOMA-IR index
(p<0.01) is ook waargeneem. Daar was ook ‘n beduidend ingekorte respons van glukose
opname deur kardiomiosiete van die vet diere na stimulasie met 10nM (p<0.05) en 100nM
(p<0.05) insulien. Disregulering van PKB/Akt is gevind in die vorm van ‘n afregulering van
die fosforilering van die proteïen (p<0.01). Die dieet het ook gelei tot die ontwikkeling van
miokardiale hipertrofie aangesien die ventrikulêre gewig (p<0.05) asook die verhouding van
die ventrikulêre gewig teenoor tibia lengte beduidend toegeneem het (p<0.01).
Eggokardiografie het ‘n toename in ventrikulêre end-diastoliese dimensie in die DIO diere
aangetoon (p<0.05). Tesame hiermee het die breedte van kardiomiosiete van die DIO diere
toegeneem (p<0.0001) en daar was ook ‘n peri-nukluêre lokalisering van NFATc3.
Behandeling van kontrole diere met ‘n GSK-3 inhibitor het insulienweerstandigheid ontlok
soos afgelei uit ‘n verhoging in liggaamsgewig (p<0.05), serum insulien-vlakke (p<0.01) en
die HOMA-IR waarde (p<0.01). In teenstelling het behandeling van die DIO diere met die
GSK-3 inhibitor tot ‘n verbetering van insulienweerstandigheid gelei aangesien ‘n verlaging
in serum insulien konsentrasies gevind is (p<0.05). Kardiomiosiete vanaf die behandelde DIO
diere het ook ‘n verhoogde insulien-gestimuleerde glukose opname met 100nM insulien
getoon (p<0.05). Behandeling met die GSK-3 inhibitor het die ontwikkeling van miokardiale hipertrofie in die
kontrole diere teweeggebring, soos aangetoon deur ‘n toename in die ventrikulêre gewig
(p<0.05) en ‘n groter selwydte in kardiomiosiete terwyl dit geen invloed op die bestaande
hipertrofie van die vet diere gehad het nie.
Gevolgtrekking: Die huidige studie het getoon dat die betrokke dieet asook behandeling met
‘n GSK-3 inhibitor insulienweerstandigheid sowel as die ontwikkelling van miokardiale
hipertrofie in rotte ontlok. In die DIO diere het die behandeling met die GSK-3 inhibitor
bloedglukose en insulien-vlakke verlaag en het nie hipertrofie vererger nie.
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