Contribution of highly active anti-retroviral therapy to the development of non-alcoholic fatty liver disease with concomitant cardiovascular dysfunction in an obese rat model

Abstract

Thesis (PhD)--Stellenbosch University, 2018.Introduction: HIV/AIDS mortality is declining due to successful highly active anti-retroviral therapy (HAART). However, obesity, non-alcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD) in treated HIV-infected populations are rising. Impaired peroxisome proliferator-activated receptor (PPARα/γ) activity is partly implicated. Aims: To assess the contribution of a protease inhibitor (Lopinavir/Ritonavir (LPV/r) and nucleoside reverse transcriptase inhibitor (Azidothymidine (AZT)/lamivudine (3TC)) HAART regimen in the development of NAFLD and CVD in rats with high caloric diet (HCD)-induced obesity, and to investigate dual PPARα/γ stimulation in limiting HAART-induced NAFLD and CVD. Methods: Wistar rats were randomised into rat chow and HCD groups (n=88/group; 16 weeks). From week 10, each group was further sub-divided into: vehicle/control, HAART, HAART+PPARα/γ agonist (Saroglitazar) and Saroglitazar only (n=22/group) administered via oral gavage. Endpoints: Daily food/water consumption, weekly total body mass (TBM), random+fasting blood glucose measurements (n=8/group); hearts exposed to either 20min global ischaemia/10min reperfusion for Western blot (WB) analysis (n=6/group) or 35min regional ischaemia/60min reperfusion (n=8/group) for haemodynamic and infarct size (IS) determinations. Liver samples were histologically assessed (n=12/group) and analysed by WB. Intraperitoneal (IP) fat was weighed. Fasting serum lipids, insulin and oxidative stress markers were measured (n=8/group). WB analyses of important signalling proteins in pre/post-cardiac ischaemia, liver and aorta tissues. Thoracic aorta (n=8/group) segments were subjected to isometric tension studies. Results: The HCD resulted in obesity (increased: TBM, %IP fat (HCD control 6.50±0.40% vs. lean control 3.60±0.3%; p<0.0001), liver mass, insulin and triglycerides (TGs). Additionally, HCD induced insulin resistance (IR). HAART+Saroglitazar led to reduced %IP fat in lean and HCD groups. HAART-induced IR, elevated cardiac mass and insulin in obese rats were limited by Saroglitazar co-treatment. HCD+HAART-induced oxidative stress (elevated conjugated dienes), was limited in combined HAART+Saroglitazar. Liver histology: HAART induced moderate hepatic steatosis in ~67% of obese rats and moderate inflammation in ~25% of cases. Combined HAART+Saroglitazar limited these changes and upregulated adenosine monophosphate-activated protein kinase (AMPK) and protein kinase B (PKB/Akt) activity, which were downregulated by HAART in HCD. Heart/aorta studies: Untreated obese rats had smaller %IS compared to lean control rats (19.1±1.6 vs. 26.1±1.6, respectively; p<0.05). IS for HCD+HAART animals were smaller (despite poor cardiac performance) compared to untreated obese rats. Post-ischaemia activity of extracellular-signal-regulated kinase (Erk1/2), PKB/Akt, AMPK and endothelial nitric oxide synthase (eNOS) was downregulated, whereas expression of p22-phox and caspase-3 was accentuated. HAART+Saroglitazar upregulated (1.5-fold) the expression of Erk1/2, PKB/Akt, AMPK and eNOS, and downregulated caspase-3 and p22-phox. Obese+HAART rats demonstrated poor aortic relaxation accompanied by downregulated eNOS and PKB/Akt, and upregulated p22-phox. However, Saroglitazar+HAART in obese animals improved aortic relaxation by ~30%, accompanied by upregulation of eNOS, and PKB/Akt, and downregulated p22-phox. Discussion and conclusion: HAART-induced NAFLD and CVD in obesity were limited by PPARα/γ agonist co-administration. HAART treatment for six weeks was not a cardiovascular risk factor per se, but it potentiated HCD-induced cardiovascular effects. Monitoring cardiovascular risk factors in obese+HAART patients is crucial, and our findings suggest that there is therapeutic potential in co-treatment with PPARα/γ agonists. The metabolic, functional and signalling disturbances in the liver, heart and aorta tissues in obese+HAART rats are interlinked, and partially limited by co-treatment with a dual PPARα/γ agonist.Inleiding: Daar is ‘n daling in die HIV/VIGS sterftesyfer a.g.v. suksesvolle hoogs-aktiewe antiretrovirale terapie (HAART). Vetsug, nie-alkoholiese vetterige lewersiekte (NAFLD) en kardiovaskulêre siekte (KVS) in behandelde HIV-geïnfekteerde populasies is aan die styg. Ingekorte peroksisoom proliferator-geaktiveerde reseptor (PPARα/γ) funksie is deels hiervoor verantwoordelik. Doelwitte: Om die bydraes van ‘n protease inhibitor (Lopinavir/Ritonavir (LPV/r) en nukleosied omgekeerde transkriptase inhibitor (Azidothymidine (AZT)/lamivudine (3TC)) as deel van HAART in die ontwikkeling van NAFLD en KVS in rotte met ’n hoë kaloriedieet (HCD)-geïnduseerde vetsug te bepaal, asook tweeledige PPARα/γ-stimulasie in die vermindering van HAART-geïnduseerde NAFLD en KVS te bepaal. Metodes: Wistar rotte is lukraak in ‘n gewone rot-dieet en ‘n HCD groep (n=88/groep; 16 weke) ingedeel. Vanaf Week 10 is elke groep verder onderverdeel in: oplosmiddel/kontrole, HAART, HAART+PPARα/γ agonis (Saroglitazar) en slegs Saroglitazar (n=22/groep) wat via ‘n gastriese buis toegedien was. Eindpunte: Daaglikse kos/water verbruik, weeklikse totale liggaamsgewig (TBM), lukrake+vastende bloedglukose (n=8/groep); harte wat blootgestel is aan óf 20min globale isgemie/10min herperfusie, wat vir Western blot (WB) ontleding aangewend is, of 35min streeksisgemie/60min herperfusie (n=8/groep), wat vir hemodinamiese en infarktgrootte (IS) bepalings aangewend is. Lewermonsters is histologies ondersoek (n=12/groep) en ontleed deur WB. Intraperitoneale (IP) vet is geweeg. Vastende serumlipiede, oksidatiewe spanningsmerkers en insulien is gemeet (n=8/groep). WB ontledings van belangrike seintransduksieproteïene in pre/post-isgemiese hart-, lewer- en aortaweefsel uitgevoer. Torakale aortasegmente (n=8/groep) is aan isometriese spanningstudies blootgestel. Resultate: Die HCD het tot vetsug gelei (verhoogde: TBM, %IP vet (HCD kontrole 6.50±0.40% vs. gewone-dieet kontrole 3.60±0.3%; p<0.0001), lewermassa, insulien en trigliseriedes). Verder het HCD tot insulienweerstandigheid (IR) gelei. HAART+Saroglitazar het tot verlaagde %IP vet in kontrole en HCD groepe gelei. HAART het IR veroorsaak, terwyl verhoogde hartmassa en insulien in vetsugtige rotte deur kobehandeling met Saroglitazar verlaag is. HCD+HAART het vrye-vetsuur-oksidasie (verhoogde gekonjugeerde diëne) geïnduseer, wat deur gekombineerde HAART+Saroglitazar verminder is. Lewerhistologie: HAART het matige lewersteatose in ~67% van vetsugtige rotte en matige inflammasie in ~25% veroorsaak. Hierdie veranderinge is deur gekombineerde HAART+Saroglitazar verlig en AMPK en PKB/Akt aktiwiteit is opgereguleer, wat deur HAART afgereguleer is. Hart/aorta studies: Onbehandelde vetsugtige rotte het kleiner IS as kontrole gehad (19.1±1.6 vs. 26.1±1.6, onderskeidelik; p<0.05). Die IS vir rotte op HAART+HCD was kleiner (ondanks swak hartfunksie) vs. hul onbehandelde eweknieë. Die post-isgemiese aktiwiteit van Erk1/2, PKB/Akt, AMPK en eNOS is afgereguleer, terwyl uitdrukking van p22-phox en kaspase-3 verhoog is. Die uitdrukking van Erk1/2, PKB/Akt, AMPK en eNOS is deur HAART+Saroglitazar opgereguleer (1.5-voudig) en PKB/Akt en p22-phox is afgereguleer. Vetsugtige+HAART rotte het swak aortiese verslapping getoon ~30%, wat met afgereguleerde eNOS en PKB/Akt en opgereguleerde p22-phox geassosieer was. Tog het Saroglitazar+HAART in vetsugtige rotte tot verbeterde aortiese verslapping, gepaard met opgereguleerde eNOS en PKB/Akt, asook afgereguleerde p22-phox gelei. Bespreking en gevolgtrekking: HAART-geïnduseerde lewersteatose en KVS in vetsugtige rotte is deur ko-behandeling met ʼn PPARα/γ agonis verlig. HAART per se was nie ‘n kardiovaskulêre risikofaktor nie, maar het die HCD-geïnduseerde kardiovaskulêre effekte vererger. Dis noodsaaklik om die kardiovaskulêre risikofaktore in vetsugtige+HAART pasiënte te monitor, en ons bevindinge toon dat ko-behandeling met PPARα/γ agoniste terapeutiese potensiaal het. Die metaboliese, funksionele en seintransduksieversteurings in die lewer, hart en aortaweefsel in vetsugtige+HAART rotte is ineengeskakel en word gedeeltelik verlaag deur ko-behandeling met ‘n tweeledige PPARα/γ agonis