Smit, Sybrand Engelbrecht (2016-03)
Rooibos is an indigenous South African plant ingested as herbal tea and well-known for its strong anti-oxidant effects. Rooibos has shown to have cardioprotective properties in vitro and in vivo, but the role of individual Rooibos flavonoids in cardioprotection still remains unclear. This in vitro study investigated Aspalathin, a dihydrochalcone unique to rooibos, for (i) cardioprotective effects in the context of age- and obesity-induced insulin resistance, known to attenuate myocardial glucose uptake and utilization and (ii) the applicable signaling pathways involved.
Male Wistar rats were allocated into three groups: 16-30 weeks feeding with either standard rat chow (C) or a high-fat, high-caloric diet (HFD), or 6-7 weeks feeding with C. Cardiomyocytes were isolated by collagenase perfusion digestion, using a Langendorff apparatus and glucose uptake determined by 2-[3H]-deoxyglucose (2DG) accumulation using liquid scinitillation analysis. In addition, H9C2 cells were differentiated into cardiomyocyte analogs and also used. Viability was tested by either Trypan-blue exclusion, JC-1-staining or PI-staining and FACS analysis, and metabolic activity determined with an ATP assay. Intracellular signaling was evaluated using Western blot analysis and commercially available antibodies to PKB and AMPK.
HFD caused significant increases in body weight gain, visceral adiposity, fasting and non-fasting blood glucose, serum insulin levels and an elevated HOMA-IR index. HFD cardiomyocytes were glucose uptake resistant to increasing concentration of insulin (1-100nM). Aspalathin (10uM) and insulin (10nM) co-incubation for 45mins induced 2DG uptake in younger control cardiomyocytes, while incubation for longer than 90 mins with aspalathin (10uM) induced 2DG uptake independent of insulin in younger control cardiomyocytes and differentiated H9C2 cells. Aspalathin improved metabolic activity and membrane integrity in cultured, differentiated H9C2 cells. Aspalathin also enhanced insulin-mediated 2DG uptake in older control cells, but failed to induce 2DG uptake in HFD cells. Acute treatment with aspalathin (15min) in conjunction with insulin in vitro significantly increased PKB activation and AMPK expression. Extended treatment with aspalathin (90mins) in young cells resulted in significantly increased AMPK activation/expression ratio, whereas aspalathin co-treatment with insulin resulted in increased PKB activation. Aged rats had significantly higher AMPK expression and activation compared to young rats. Conclusions:
A high-fat, high-sucrose diet of at least 16 weeks is an effective model to induce insulin-resistant, obese rats. Aspalathin and insulin co-treatment for 45 mins in cardiomyocytes isolated from young rats, and co-treatment for 90 mins in aged, control rats, induced glucose uptake. Aspalathin of at least 90 mins induced glucose uptake in cardiomyocytes from young Wistar rats, and differentiated H9C2 cells. In addition, it resensitized the insulin-signaling pathway in cardiomyocytes, possibly through activation of PKB and AMPK, resulting in an additive response. These beneficial effects of aspalathin may ultimately be due to its antioxidant capacity, receptor-mediated actions or role in GLUT4 translocation, but this remains to be established.
Rooibos is ‘n inheemse plant, eie aan Suid-Afrika, wat as tee gedrink word en bekend is vir sy sterk anti-oksidant eienskappe. Rooibos speel ‘n belowende rol in die beskerming van die hart, maar die rol van sy individuele flavanoïdes moet steeds ondersoek word in die konteks van die hart. In hierdie in vitro studie is die rol van aspalatien, ‘n ‘dihidrochalcone’ uniek aan rooibos, ondersoek vir sy: (i) vermoë om die hart te beskerm in die konteks van ouderdom- en vetsug-geïnduseerde insulienweerstandigheid (albei risiko faktore wat glukose opname deur die hartspiere verswak), en (ii) die moontlike seintransduksiepaaie betrokke tydens hierdie proses.
Manlike Wistar rotte is ingedeel in drie groepe: ‘n 16-30 weke dieet met standaard rotkos (C), ‘n hoë vet, hoë koolhidraat dieet vir 16-30 weke (HFD), of ‘n 6-7 weke standaard rotkos groep. Kardiomiosiete is deur kollagenase perfusievertering geïsoleer deur gebruik te maak van ‘n Langendorff sisteem, en daarna is die glukose opname bepaal deur 2-[3H]-deoksiglukose (2DG) akkumulasie te meet. Verder is H9C2 selle gedifferensieer om hartsel analoë te vorm en ook bestudeer. Sellewensvatbaarheid is bepaal deur Trypan-blou eksklusie, JC-1-kleuring of propidium jodied-kleuring en vloeisitometrie en die metaboliese aktiwiteit is bepaal deur ATP vlakke te meet. Intrasellulêre seinstransduksie is ondersoek deur Western klad analise en die kommersiëel-beskikbare teenliggaampies teen PKB and AMPK.
HFD het beduidende verhogings in liggaamsgewig, intraperitoneale vet, vastende- en nie-fastende bloed glukose, serum insulienvlakke en die HOMA-IR indeks tot volg gehad. Verder was die glukose opname van HFD hartselle onderdruk in reaksie op stapsgewyse verhogings in insulin konsentrasies (1-100nM). In jong rothartselle kon ‘n kombinasie van aspalatien (10uM) en insulin (10nM) vir 45min 2DG opname verhoog, terwyl ten minste 90min van aspalatien behandeling nodig was om 2DG opname in jong rot hartselle en gedifferensieerde H9C2 selle te verhoog. Verder het 1uM aspalatien ook die metaboliese aktiwiteit en membraanintegriteit van gedifferensieerde H9C2 selle in kultuur verhoog. Aspalatien kon insulien-afhanklike 2DG opname in hartselle van C diere verhoog, slegs na 90 minute in kombinasie met insulien. Aspalatien het geen effek gehad as behandeling op HFD selle nie. Akute behandeling met 10nM insulien in kombinasie met 10uM aspalatien (15mins) in vitro het ‘n verhoogde PKB aktivering en AMPK uitdrukking tot volg gehad. Verlengde behandeling met aspalatien (90mins) het gelei tot ‘n beduidende verhoging in die AMPK aktiverings-/uitdrukking verhouding, terwyl kombinasie met insulien PKB beduidend geaktiveer het. Ouer rotte het beduidend hoër vlakke en aktivering van AMPK as jonger rotte gehad.
‘n Hoë-vet, hoë-sukrose dieet van ten minste 16 weke is voldoende om insulienweerstandigheid en vetsug te induseer in ‘n Wistar rot model. Aspalatien kan insulien-afhanklike glukose opname verhoog na 45 minute behandeling in jong rothart selle, terwyl ten minste 90 minute benodig word om dieselfde effek in ouer kontrole rotte te verkry. Aspalatien het glukose opname in die hartselle van jong Wistar rotte, asook gedifferensieerde H9C2 selle ontlok na ten minste 90 minute van behandeling. Verder het aspalatien die insulien-seinstransduksie pad in hartselle gesensitiseer, heel moontlik deur aktivering van PKB and AMPK wat lei tot ‘n verhoogde glukose opname in kombinasie met insulien. Hierdie voordelige effekte van aspalatien kan heel moontlik aan sy antioksidant kapasiteit toegeskryf word, asook die spesifieke reseptor-gemedieerde effekte, en/of sy rol in GLUT4 translokering, maar dit moet nog bevestig word.
Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/98490