Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Introduction
Diabetes constitutes a major health challenge. Since cardiovascular complications are common in
diabetic patients this will further increase the overall burden of disease. Furthermore, stress-induced
hyperglycemia in non-diabetic patients with acute myocardial infarction is associated with higher inhospital
mortality. Hyperglycemia-induced oxidative stress results in DNA damage and subsequent
activation of poly-ADP-ribose polymerase (PARP) as a restorative mechanism. However, PARP
attenuates glyceraldehyde–3-phosphate dehydrogenase (GAPDH) activity, thereby diverting upstream
glycolytic metabolites into damaging non-oxidative glucose pathways (NOGP). For example,
hyperglycemia-induced stimulation of four NOGP, i.e. the polyol pathway, hexosamine biosynthetic
pathway (HBP), advanced glycation end products (AGE), and PKC activation elicit cardiovascular
complications. The current thesis examined the regulation of NOGP in the setting of ischemia and
reperfusion under hyperglycemic conditions.
Here we hypothesized that administration of two unique therapeutic interventions, i.e. oleanolic acid
(OA; clove extract) and benfotiamine (BFT; vitamin B1 derivative), can blunt oxidative stress and
NOGP-induced cardiac dysfunction under hyperglycemic conditions following ischemia and
reperfusion. Our choice for these agents was based on the principle that OA possesses antioxidant
properties; and BFT stimulates transketolase (pentose phosphate pathway [PPP] enzyme) thereby
shunting flux away from the NOGP pathways. Additionally, hyperglycemia-induced oxidative stress
can also result in dysregulation of the ubiquitin-proteasome system (UPS) that removes misfolded
proteins. There are conflicting data whether increased/decreased UPS is detrimental with
hyperglycemia and/or in response to ischemia and reperfusion. In light of this, we also hypothesized
that BFT and OA act as novel cardio-protective agents by diminishing myocardial UPS activity in
response to ischemia and reperfusion under acute hyperglycemic conditions.
Materials and Methods
For the first part of the study, we employed several experimental systems: 1) H9c2 cardiac myoblasts
were exposed to 33 mM glucose for 48 hr vs. controls (5 mM glucose); and subsequently treated with
two OA doses (20 and 50 μM) for 6 and 24 hr, respectively; 2) Isolated rat hearts were perfused ex
vivo with Krebs-Henseleit buffer containing 33 mM glucose vs. controls (11 mM glucose) for 60 min,
followed by 20 min global ischemia and 60 min reperfusion ± OA treatment; 3) Infarct size was determined using Evans Blue dye and 1% 2,3,5-triphenyl tetrazolium chloride (TTC) staining with 20
min regional ischemia and 2 hr reperfusion 4) In vivo coronary ligations were performed on
streptozotocin-diabetic rats ± 0.45 mg/kg OA administration within the first two minutes of reperfusion;
and 5) Effects of long-term OA treatment (2 weeks) on heart function were assessed in streptozotocin
(STZ)-diabetic rats. Here, STZ was dissolved in citrate buffer (p.H 6.3) and diabetes was induced by
administering 60 mg/kg i.p Tissues were collected at the end of the global ischemia experiments and
analyzed for oxidative stress, apoptosis, UPS activity and HBP activation.
For the second part of the study we employed several experimental systems: 1) Isolated rat hearts
were perfused ex vivo with Krebs-Henseleit buffer containing 33 mM glucose vs. controls (11 mM
glucose) for 90 min, followed by 30 min global ischemia and 60 min reperfusion ± 25, 50 and 100 μM
BFT treatment, respectively, added during the first 20 min of reperfusion; 2) Infarct size determination
as in #3 above but with 30 min regional ischemia and 2 hr reperfusion ± 100 μM BFT treatment; and 3)
In vivo coronary ligations performed on streptozotocin-diabetic rats ± 0.50 mg/kg BFT treatment within
the first two min of reperfusion. In parallel experiments, NOGP inhibitors were added during the first 20
min of reperfusion. The following inhibitors were individually employed: AGE pathway (100 μM
aminoguanidine); PKC (5 μM chelerythrine chloride); HBP (40 μM 6-diazo-5-oxo-L-norleucine); and
polyol pathway (1 μM zopolrestat); Infarct size determination as in #2) with 30 min regional ischemia
and 120 min reperfusion ± similar treatments.
Results
Our data show decreased cardiac contractile function in response to ischemia and reperfusion under
hyperglycemic conditions. This was linked to increased PARP and attenuated GAPDH activities,
together with higher activation of the NOGP. Moreover, we found elevated myocardial oxidative stress,
UPS and cell death under these conditions. OA treatment resulted in cardio-protection, i.e. for ex vivo
and in vivo rat hearts exposed to ischemia and reperfusion under hyperglycemic conditions. In
parallel, OA decreased oxidative stress, apoptosis, HBP flux and UPS activity following ischemia and
reperfusion. Long-term OA treatment also improved heart function in streptozotocin-diabetic rats. Our
data also reveal that acute BFT treatment significantly decreased myocardial oxidative stress and
apoptosis, and provided cardio-protection in response to ischemia and reperfusion under
hyperglycemic conditions. In parallel, BFT blunted hyperglycemia-induced activation of four NOGP in
the rat heart. Acute administration of each of the NOGP inhibitors decreased PARP and enhanced GAPDH
activities, while diminishing oxidative stress and myocardial apoptosis. Moreover, each of the NOGP
inhibitors (individually) employed blunted activation of the other three pathways here examined. Hearts
treated with NOGP inhibitors also displayed improved functional recovery and smaller infarct sizes
following ischemia and reperfusion. Interestingly, NOGP inhibitors resulted in the same degree of
change (for all above-mentioned parameters evaluated) when compared to each other.
Conclusions
This study shows that acute and chronic hyperglycemia trigger myocardial oxidative stress that
eventually results in NOGP activation and contractile dysfunction following ischemia and reperfusion.
Moreover, our findings establish - for the first time as far as we are aware - that there is a convergence
of downstream NOGP effects in our model, i.e. increased myocardial oxidative stress, further NOGP
pathway activation, apoptosis, and impaired contractile function. Thus a vicious metabolic cycle is
established whereby hyperglycemia-induced NOGP further fuels its own activation by generating even
more oxidative stress, thereby exacerbating damaging effects on the heart under these conditions. We
also found that both OA and BFT treatment blunted high glucose-induced detrimental effects and
provided robust cardio-protection in response to ischemia and reperfusion under hyperglycemic
conditions (acute and chronic). These findings suggest that the UPS may be a unique therapeutic
target to treat ischemic heart disease in individuals that present with stress-induced, acute
hyperglycemia. Moreover, BFT exhibited its cardio-protective effects by NOGP inhibition after
ischemia and reperfusion under acute and chronic high glucose conditions. A similar effect was
observed at baseline although the underlying mechanisms driving this process still need to be
elucidated. In summary, the findings of this thesis are highly promising since it may eventually result in
novel, cost-effective therapeutic interventions to treat acute hyperglycemia (in non-diabetic patients)
and diabetic patients with associated cardiovascular complications. / AFRIKAANSE OPSOMMING: Inleiding
Diabetes skep ‘n groot gesondheidsuitdaging. Omrede kardiovaskulêre komplikaseis algemeen onder
diabetiese pasiënte is, sal dit oorkoepelend die las van hierdie siekte verder laat toeneem. Verder
word stresgeïnduseerde hiperglukemie in nie-diabetiese pasiënte met akute miokardiale infarksie
geassosieër met ‘n hoër binne-hospitaalmortaliteit. Hiperglukemies-geïnduseerde oksidatiewe stres
veroorsaak DNA skade, en gevolglike aktivering van poli-ADF-ribose polimerase (PARP), as ‘n
herstelmeganisme. Nietemin, PARP verminder gliseraldehied–3-fosfaatdehidrogenase (GAPDH)
aktiwiteit om sodoende die opstroom glikolitiese metaboliete te herlei na skadelike nie-oksidatiewe
glukose weë (NOGW). Byvoorbeeld, hiperglukemie-geïnduseerde stimulasie van vier NOGW, i.e. die
poliolweg, heksosamienbiosintetiese weg, (HBW), gevorderde glukasie eindprodukte (GGE), en PKC
aktivering, lei tot kardiovaskulêre komplikasies. Die huidige tesis ondersoek die regulering van NOGW
in ‘n isgemiese-reperfussie onder hiperglukemiese toestande.
Ons hipotetiseer dat die toediening van twee unieke terapeutises intervensies, i.e. oleanoliese suur
(OS, naaltjie ekstrak), en benfotiamien (BFT, vitamien B1 derivaat) oksidatiewe stress kan versag, en
NOGW geinduseerde kardiale disfunksie onder hiperglukemiese toestande na ischemie en
reperfussie. Ons keuse vir hierdie middels is gebaseer op die beginsel dat OS antioksidanteienskappe
bevat, en dat BFT transketolase (pentosefosfaat weg (PFW) ensiem) stimuleer en
sodoende die fluks weg van die NOGW weg veroorsaak. Addisioneel kan hiperglukemiegeïnduseerde
oksidatiewe stres ook tot wanregulering van die ubikwitien-proteosoomsisteem (UPS)
wat wangevoude protïene verwyder, aanleiding gee. Daar bestaan kontrasterende data oor ‘n
verhoogde/verlaagde UPS, tesame met hiperglukemie en/of in reaksie tot isgemie-reperfussie. In die
lig hiervan, hipotetiseer ons dat BFT en OS as ‘n nuwe kardiobeskermingsmiddel kan optree deur
miokardiale oksidatiewe stres en UPS aktiwiteit in reaksie op isgemie-reperfussie tydens akute
hiperglukemiese toestande kan verlaag.
Materiale en Metodes
Vir die eerste deel van die studie het ons van verskeie eksperimentele sisteme gebruik gemaak: 1)
H9c2 kardiale mioblaste is aan 33 mM glukose vir 48 uur vs. kontrole (5 mM glukose) blootgestel; en
gevolglik met twee OS dosisse (20 en 50 μM) vir 6 en 24 hr, onderskeidelik behandel; 2) geïsoleerde
rotharte is ex vivo met Krebs-Henseleit buffer, wat, 33 mM glukose vs. kontrole (11 mM glukose) bevat, vir 60 min geperfuseer, daarna is dit deur 20 min globale isgemie gevolg en 60 min reperfussie
± OS behandeling; 3) Infarkgrootte is bepaal deur Evans bou kleursel en 1% 2. 3-5 tripfeniel
tetrazoloimcholierd (TTC) kleuring met 20 minute regionale ischemie, en 2 uur reprefussie 4) In vivo
koronêre liggasies is op streptozotosien-diabetiese rotte uitgevoer ± 0.45 mg/kg OS toedienning binne
die eerste twee minute van reperfussie; en 5) effekte van langtermyn OS behandeling (2 weke) op
hartfunskie is in hierdie streptozotosien-diabetiese rotte ondersoek. Hier is STZ opgelos in ‘n
sitraatbuffer (pH 6.3), en diabetes is geinduseer deur 60mg/kg i.p. toe te dien. Weefsels is aan die
einde van die globale isgemie eksperimente versamel, en vir oksidatewe stres, apoptose, UPS
aktiwiteit en HBW aktivering, ontleed.
Vir die tweede deel van die studie het ons van verskeie eksperimentele sisteme gebruik gemaak: 1)
geïsoleerde rotharte is ex vivo met Krebs-Henseleit buffer, wat 33 mM glukose vs. kontrole (11 mM
glukose) bevat, vir 90 min geperfuseer. Daarna is dit gevolg met 30 min globale isgemie en 60 min
reperfussie ± 25, 50 en 100 μM BFT behandeling onderskeidelik, gevolg, bykomend, gedurende die
eerste 20 min reperfussie; 2) Infarkgrootte is bepaal soos in #3 hierbo, maar met 30 minute regionale
ischemie en 2 uur reperfussie ± 100 μM BFT behandeling; en 3) In vivo koronêre liggasies is op
streptozotosien-diabetiese rotte uitgevoer ± 0.50 mg/kg BFT behandeling binne die eerste twee
minute van reperfussie. Met parallele eksperimente is NOGW inhibeerders bygevoeg binne die eerste
20 min van reperfussie. Die volgende inhibeerders is individueel ontplooi: GGE weg (100 μM
aminoguanidien); PKC (5 μM chelleritrienchloried); HBW (40 μM 6-diazo-5-oxo-L-nor-leusien); en
poliolweg (1 μM zopolrestaat); 2) Infarkgrootte is bepaal soos in #2) met die uitsondering van 30 min
regionale isgemie en 120 min reperfussie ± identiese behandelings.
Resultate
Ons data toon aan dat kardiale kontraktiele funksie, in reaksie op isgemie-reperfussie onder
hiperglukemiese toestande, verlaag. Dit is verwant aan verhoogde PARP en verminderde GAPDH
aktiwiteit, tesame met ‘n hoër aktivering van die NOGW. Verder het ons bevind dat verhoogde
miokardiale oksidatiewe stres, UPS en seldood onder die toestande voorkom. OS behandeling lei tot
kardiale beskerming, i.e. vir ex vivo en in vivo rotharte wat aan isgemie-reperfussie onder
hiperglukemiese toestande blootgestel is. Parallel hiermee het OS oksidatiewe stres, apoptose, HBW
invloed, en UPS aktiwiteit na isgemie-reperfussie, verlaag. Langtermyn OS behandeling het ook
hartfunksie in streptozotosien-diabetiese rotte verbeter. Ons data vertoon verder dat akute BFT
behandeling, miokardiale oksidatiewe stres en apoptose, betekenisvol verlaag het in reaksie op isgemie-reperfussie onder hiperglukemiese toestande. Parallel hiermee het BFT hiperglukemiegeïnduseerde
aktivering van vier NOGWë in die rothart, verminder.
Akute toediening van die elk van die NOGW inhibeerders het PARP verlaag, en GAPDH aktiwiteite
verhoog, terwyl oksidatiewe stres, en miokardiale apoptose verminder. Verder het elk van die NOGW
inhibeerders wat (individueel) toegedien is, aktivering van die ander drie weë, hier ondersoek, verlaag.
Die harte wat met NOGW inhibeerders behandel is het ook ‘n verbeterde herstel en kleiner
infarkgrootte na isgemie-reperfussie getoon. Interessant is hoe die NOGW inhibeerders tot dieselfde
graad verandering (vir al die bogemelde parameters wat geevalueer is) indien dit vergelyk word teen
mekaar, gelei het.
Gevolgtrekking
Hierdie studie het bevind dat akute en chroniese hiperglukemie, miokardiale oksidatiewe stres ontlok,
en dat dit geleidelik tot NOGW aktivering en kontraktiele wanfunksionering na isgemie-reperfussie lei.
Verder het ons bevindinge vir die eerste keer, volgens ons wete, bewys dat daar ‘n ineenloping is van
afstroom NOGW effekte in ons model, i.e. verhoogde miokardiale oksidatiewe stres, verdere NOGW
weg aktivering, apoptose, en ingeperkte kontraktiele funksie. Dus, ‘n gebrekkige metaboliese siklus
word verkry waarby hiperglukemies-geïnduseerde NOGW verder sy eie aktivering aanvuur deur meer
oksidatiewe stres, en sodoende die skadelike effekte op die hart onder hierdie toestande verder
versleg. Ons het verder bevind dat beide OS en BFT behandeling, hoë glukose-geïnduseerde
skadelike effekte onderdruk, en kragtige kardiale-beskerming in reaksie op isgemie-reperfussie onder
hiperglukemiese toestande (akuut en chronies), teweeg bring. Hierdie bevindinge dui moontlik daarop
dat die UPS ‘n unieke terapeutiese teiken kan wees vir die behandeling van isgemiese hartsiekte in
individue wat presenteer met stres-geïnduseerde, akute hiperglukemie. BFT het ook sy kardiale
beskermende effekte getoon deur NOGW inhibering na isgemie-geïnduseerde reperfussie onder
aktute en chroniese hoë glukose toestande. ‘n Soorgelyke effek is tydens die basislyn waargeneem,
alhoewel die onderliggende meganisme wat hierdie proses dryf verder ondersoek moet word.
Opsommend is ons bevindinge baie belowend omrede dit daartoe kan aanleiding gee tot ‘n nuwe,
meer koste-effektiewe terapeutiese intervensie vir die behandeling van akute hiperglukemie (in niediabetiese pasiënte) en diabetiese pasiënte met geassosieërde kardiovaskulêre komplikasies. / Oppenheimer, Beit Trust and Harry Crossley
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/79900 |
| Date | 03 1900 |
| Creators | Mapanga, Rudo Fiona |
| Contributors | Essop, M. Faadiel, Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xxxv, 336 p. : ill. |
| Rights | Stellenbosch University |
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