TKTL1 plays a critical role in ameliorating intracellular oxidative stress under hyperglycemic conditions

Reuter, Sulandy (2018-03)

Thesis (MSc)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: Diabetes is a growing, global concern, reaching a prevalence far beyond the projected statistics. Hyperglycemia is a common denominator of type 1 and type 2 diabetes mellitus that if left untreated, results in detrimental outcomes, e.g. oxidative damage. The pentose phosphate pathway (glucose-6-phosphate dehydrogenase [G6PD] and transketolase [TKT] as rate-limiting enzymes) offers an alternate ‟safer” route for excess metabolic fuels, while also replenishing anti-oxidant defenses and thus potentially offers value as a novel therapeutic target. As TKT-like 1 (TKTL1), a relatively novel member of the transketolase family, has been overlooked in this regard, we employed a TKTL1-knock-out (KO) mouse model as a targeted approach to evaluating TKTL1 function. Multiple low doses of streptozotocin (STZ) (40 mg/kg, intraperitoneal injections on five consecutive days) were used to induce hyperglycemia, a pathophysiologic state known to culminate in oxidative stress and damaging outcomes. Hyperglycemia incidence was 40% and 90% in wild-type (WT) and TKTL1-KO STZ-injected male groups, respectively, while only the TKTL1-KO STZ group displayed significantly higher blood glucose levels compared to the WT control (p<0.001) and the TKTL1-KO control group (p<0.001). The male TKTL1-KO STZ-treated group displayed larger relative liver weights (p<0.001) and kidney weights (p<0.05), respectively, compared to both WT and TKTL1-KO controls. Gastrocnemii weight of the TKTL1-KO STZ-treated group was lower than that of the WT control group (p<0.01). There was a significant, negative correlation between liver G6PD activity and blood glucose levels (Spearman’s correlation coefficient [r]=-0.37, p=0.03) while this correlation was not observed in cardiac tissue. In the latter, both TKTL1-KO groups displayed ~50% diminished transketolase activity compared to the WT control (p<0.001). No significant differences were noted in any of the oxidative stress and damage assessments of blood, heart, or liver samples. However, several adaptations were revealed in hepatic anti oxidant defenses: STZ-treated TKTL1-KO mice displayed increased catalase activity (p<0.05), with catalase activity showing a significant, positive correlation with blood glucose (r=0.39, p=0.02); ferric reducing anti-oxidant power (FRAP) concentrations as well as the reduced glutathione (GSH):to oxidized glutathione (GSSG) ratio were, significantly decreased in STZ-treated TKTL1-KO group (p<0.05) while both of these measurements were negatively correlation with blood glucose levels (r=0.45, p<0.01 for FRAP, r=-0.36, p=0.03 for GSH:GSSG ratio). Our data reveal that TKTL1 may play an anti-oxidant role in the liver to protect against hyperglycemia and to maintain overall glucose homeostasis. Thus our study demonstrates novel insights into TKTL1 function and shows that it remains a feasible therapeutic target for the treatment of diabetes.

AFRIKAANSE OPSOMMING: Diabetes is ‘n wêreldwye bekommernis waarvan die geprojekteerde voorkoms by verre onderskat is. ‘n Algemene kenmerk van tipe 1 sowel as tipe 2 diabetes mellitus is hiperglukemie wat lei tot skadelike uitkomste soos oksidatiewe skade. Die pentose-fosfaat padweg (PPP) (glukose-6-fosfaat dehidrogenase [G6PD] en transketolase [TKT] as tempo aangewende ensieme) bied ‘n alternatiewe, ‟veiliger” weg vir oorskot metaboliese brandstof terwyl dit ook antioksidante aanvul, dus is die PPP ‘n potensiële teiken vir behandeling. Siende dat ‘n ensiem soortgelyk aan TKT, TKTL1, se potensiaal in hierdie area van navorsing nog nie ontgin is nie het ons ‘n TKTL1-uitslaan (KO) muismodel gebruik as ‘n geteikende benadering om die funksie van TKTL1 te evalueer. Lae dosise van streptozotosien (STZ) (40 mg/kg daagliks op vyf agtereenvolgende dae) was gebruik om hiperglukemie, ‘n patologie geassosieer met oksidatiewe stress en skadelike uitkomstes, te bewerkstellig. Hiperglukemie voorkoms in die manlike STZ-behandelde groepe was 40% in wilde-tipe (WT) en 90% in die TKTL1-KO, waar slegs die TKTL1-KO groep wat STZ behandeling ontvang het, beduidend hoër bloedglukosevlakke getoon het in vergelyking met die WT kontrole groep (p<0.001). Die manlike TKTL1-KO STZ-behandelde groep se lewer- (p<0.001) en nier gewig (p<0.05) was verhoog in vergelyking met beide die WT en die TKTL1-KO kontroles. Inteenstelling hiermee, was die gastrocnemi spiere van die TKTL1-KO groep se gewig kleiner as dié van die WT kontrole groep (p<0.01). ‘n Beduidende, negatiewe korrelasie is opgemerk tussen lewer G6PD aktiwiteit en bloedglukosevlakke (Spearman se korrelasie koëffisiënt [r] =-0.37, p=0.03), wat nie in die hart van toepassing was nie. In hartweefsel het beide TKTL1-KO groepe ‘n ~50% verlaging getoon in TKT aktiwiteit in vergelyking met die WT kontrole (p<0.001). Geen beduidende veranderige in oksidatiewe stres en –skade asseseerings is gevind in bloed, hartweefsel of lewerweefsel nie. Hierinteen is verskeie aanpassings in die antioksidatiewe verdedigings meganismes opgelet. STZ-behandelde TKTL1-KO muise het verhoogde katalase aktiwiteit getoon (p,0.05) terwyl ‘n positiewe korrelasie ook opgemerk is tussen katalase aktiwiteit en bloedglukose vlakke (r=0.39, p=0.02); yster-reduserende antioksidant mag (FRAP) asook die fraksie van gereduseerde glutathione (GSH):geoksideerde glutathione (GSSG) was albei beduidend verlaagin die STZ-behandelde TKTL1-KO groep (p<0.05) terwyl beide van hierdie merkers ‘n negatiewe korrelasie met bloedglukosevlakke getoon het (r=0.45, p<0.01 vir FRAP, r=-0.36, p=0.03 vir GSH:GSSG fraksie). Ons data onthul ‘n antioksidatiewe rol vir hierdie ensiem in die lewer wat beskerm teen hiperglukemie en selfs kan byrae tot algehele glukose homeostase. Dus lewer ons studie nuwe insigte in die funksie van TKTL1 wat steeds ‘n moontlike teiken is vir die behandeling van diabetes.

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