The effect of chronic ingestion of afriplex grt™ on myocardial insulin resistance and mitochondrial function – a preclinical study.

Kroukamp, Marlouw (2018-03)

Thesis (MScMedSc)--Stellenbosch University, 2018.

Thesis

Introduction: Obesity is an excessive fat accumulation in the body, known to be a significant precursor to insulin resistance and type 2 diabetes. Complications associated with obesity can contribute to the development of cardiovascular disease which, in turn, is associated with abnormal mitochondrial energetics. Rooibos (Aspalathus linearis) has known antidiabetic, anti-obesity and cardiovascular benefits. We investigated these properties with a focus on mitochondrial function, using an aspalathin-rich green rooibos extract, Afriplex GRT Extract. Aims: (i) To determine whether Afriplex GRT Extract ingestion can improve the insulin resistance state elicited by a high fat diet using a rat model. (ii) To determine whether Afriplex GRT Extract ingestion has any effects on myocardial mitochondrial oxidative phosphorylation potential and the process of mitophagy Methods: Male Wistar rats were fed either a control or a high fat diet (HFD) for 16 weeks. Half of each group was treated with Afriplex GRT Extract (60mg/kg/day) from weeks 11 to 16. Body weight, food and water intake were monitored, and liver- and intraperitoneal fat (IP) weighed at sacrifice. After sacrifice, myocardial mitochondria were isolated. Half of these were used to measure respiration on an oxygraph and the other half for Western Blot analysis of proteins involved in mitophagy. In addition, hearts were freeze clamped immediately after sacrifice and used to determine levels of insulin signalling proteins. Results: The HFD caused increased body weight-, liver- and IP fat gain, and elevated leptin levels while it decreased insulin signalling protein expression (GLUT4, tAkt/PKB and tAMPK). Ingestion of Afriplex GRT Extract diminished the weight gain, liver weight gain, IP fat weight gain and leptin in HFD animals. Moreover, it improved oral glucose tolerance in control animals and, according to a borderline significance in a HOMA-IR calculation, insulin resistance in the control animals. However, an increased basal glucose level was seen in HFD animals treated with Afriplex GRT Extract. Afriplex GRT Extract improved mitochondrial coupling efficiency in HFD animals and oxidative phosphorylation in control animals. Afriplex GRT Extract decreased mitochondrial p62 and cytosolic Beclin-1 levels and BNIP3 dimerization in the mitochondria of the control animals, while increasing autophagic flux (LC3-II expression) in hearts from HFD animals. A decrease in Sesn2 levels indicated possible anti-oxidant effects. Conclusion: We conclude that Afriplex GRT Extract resulted in less weight gain in HFD animals, improved whole-body insulin sensitivity and enhanced mitochondrial function. At the dose used, no detrimental effects were observed.

Inleiding: Vetsug is ‘n oormatige vet akkumulasie in die liggaam en ‘n belangrike oorsaaklike faktor vir insulien-weerstandigheid en tipe 2 diabetes. Komplikasies geassosieer met vetsug kan bydra tot die ontwikkeling van kardiovaskulêre siektes, wat weer geassosieer word met abnormale mitochondriale funksie. Rooibos (Aspalathus linearis) is bekend vir anti-diabetiese, anti-vetsug en kardiovaskulêre voordele. Ons het hierdie eienskappe ondersoek met ‘n fokus op mitochondriale funksie, deur die gebruik van ‘n aspalatien-ryk groen rooibos ekstrak, Afriplex GRT Ekstrak. Doelstellings: (i) Om te bepaal of die inname van Afriplex GRT Ekstrak insulien-weerstandigheid, geïnduseer deur ‘n hoë vet dieet in ‘n rotmodel, kan verbeter. (ii) Om te bepaal of die inname van Afriplex GRT Ekstrak enige effek het op miokardiale mitochondrale oksidatiewe fosforilasie potensiaal en die proses van mitofagie. Metodes: Manlike Wistar rotte is gevoer met óf ‘n normale kontrole dieët (standaard rotkos) óf ‘n hoë vet dieët (HFD) vir 16 weke. Die helfte van elke groep is behandel met Afriplex GRT Ekstrak (60mg/kg/dag) vanaf weke 11 tot 16. Liggaamsgewig, kos- en water inname was gemonitor en die lewergewig en intra-peritoniele (IP) vet gemeet na terminasie. Miokardiale mitochondria is geisoleer: een helfte van die preparaat was gebruik om respirasie te meet in ‘n oksigraaf, en die ander helfte vir Westerse klad analise van mitofagie geassosieerde proteïene. Harte is ook onder basale omstandighede gevriesklamp en gebruik om die vlakke van die insulien seintransduksiepad proteïene te bepaal. Resultate: Die HFD het ‘n verhoging veroorsaak in liggaamsgewig-, lewer- en IP vet toename, sowel as in leptien vlakke teweeggebring. Daar was ook ‘n afname in insulienseintransduksie proteïene (GLUT4, tAkt/PKB, en tAMPK). Die inname van Afriplex GRT Ekstrak het die gewig toename sowel as die lewer, IP vet toename en leptien vlakke in die HFD diere verlaag. Afriplex GRT Ekstrak het die orale glukose toleransie in kontrole diere en, volgens ‘n grensgeval beduidendheid van ‘n HOMA-IR berekening, insulien weerstandigheid in die kontrole diere verbeter. Daarteenoor is verhoogde basale glukose vlakke in die HFD diere wat behandel was met Afriplex GRT Ekstrak, waargeneem. Afriplex GRT Ekstrak behandeling het die mitochondriale koppeling in die HFD diere en oksidatiewe fosforilasie in die kontrole diere verbeter. Dit het ook ‘n afname veroorsaak in mitochondriale p62, sitosoliese Beclin-1 vlakke en BNIP3 dimerisering in die harte van kontrole diere, terwyl dit ‘n verhoogte autofagie-fluks (LC3-II uitdrukking) in die harte van die HFD groepe veroorsaak het. Verlaagde Sesn2 uitdrukking dui moontlike anti-oksidatiewe effekte aan. Gevolgtrekking: Afriplex GRT Ekstrak het gewigsverlies in HFD diere teweeggebring en insulien-weerstandigheid en mitochondriale funksie verbeter. Met die dosis gebruik was daar geen nadelige effekte waargeneem nie.

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