The effect of an aspalathin-rich rooibos extract on inflammatory crosstalk between adipocytes and muscle cells

Obonye, Nnini (2021-12)

Thesis (MSc)--Stellenbosch University, 2021

Thesis

ENGLISH ABSTRACT: The skeletal muscle not only plays a role in maintaining posture and generating force (contraction), but also is a dynamic metabolically active tissue that plays a central role in the regulation of glucose metabolism. Western diets and a sedentary lifestyle reduce the contribution of skeletal muscle to the regulation of blood glucose. In addition, inflammation resulting from chronic elevated levels of pro-inflammatory cytokines is implicated in the development of insulin resistance, metabolic disease and loss of skeletal muscle mass. The gut plays a central role in the maintenance of metabolic health and disease. Metabolic disease is associated with gut dysbiosis and “leaky” gut syndrome releasing lipopolysaccharide (LPS) into the circulation driving a chronic proinflammatory cytokine response. To date there have been numerous studies demonstrating that Aspalathus linearis (rooibos) plant polyphenols protect against the development of metabolic diseases. The aim of this study was three-fold. Firstly, study the effects of LPS on skeletal muscle growth and metabolism in vitro using murine C2C12 myoblasts. Secondly, to study the effect(s) of 3T3-L1 adipocyte derived adipokines on LPS-induced pro-inflammatory cytokine secretion by myoblasts, and thirdly, to investigate the effect of an aspalathin-rich green rooibos extract (Afriplex GRT™) on the LPSinduced immune responses. To establish the skeletal muscle model of inflammation, C2C12 myoblasts were exposed to LPS (0.1 μg/mL and 1 μg/mL) for 24 hours and treated with Afriplex GRTTM (GRT) (1 μg/mL and 10 μg/mL) for 24 hours. Cell viability, inflammation (IL-6 secretion), glucose uptake, and expression of relevant genes and proteins were assessed. Furthermore, C2C12 myoblasts were differentiated in the presence of LPS to assess the effects of inflammation on myogenesis. A coculture system using C2C12 myoblasts and 3T3-L1 pre-adipocytes and mature adipocytes were used to study the secretion of IL-6 and adiponectin. LPS was a potent inducer of pro-inflammatory IL-6 cytokine response, specifically in myoblasts as opposed to myotubules, suggesting that IL-6 is differentially regulated in myotubules. Myoblasts exposed to LPS during differentiation results in decreased myotube width and number suggesting that metabolic endotoxemia affects muscle mass and potentially affecting skeletal muscle energy metabolism. The myogenic regulatory factors, myogenin, MyoD and myostatin were downregulated by LPS during myoblast differentiation. In co-culture, LPS significantly increased IL-6 secretion in both myoblasts and 3T3-L1 pre-adipocytes, whereas IL-6 secretion was modulated by the differentiated 3T3-L1 adipocytes in co-culture. A dramatic increase in adiponectin levels secreted by the differentiated 3T3-L1 adipocytes compared to the preadipocytes could have accounted for the lower IL-6 secretion in the co-culture. GRT was unable to ameliorate the effects of LPS-induced inflammation.

AFRIKAANS OPSOMMING: Die skeletspier speel nie slegs 'n rol met liggaamshouding en die lewering van krag (sametrekking) nie, maar is ook 'n dinamiese metabolies-aktiewe weefsel wat 'n sentrale rol in die regulering van glukose-metabolisme speel. Westerse diëte en 'n onaktiewe lewenstyl verminder skeletspiere se vermoë om bloedglukose vlakke te help reguleer. Ook is inflammasie, as gevolg van chroniese verhoogde vlakke van pro-inflammatoriese sitokiene, betrokke by die ontwikkeling van insulienweerstandigheid, metaboliese siektes en verlies aan skeletspiermassa. Die dermkanaal speel 'n sentrale rol in die instandhouding van metaboliese gesondheid en siektes. Die 'lekkende' dermsindroom, wat met metaboliese siekte geassosieer word, veroorsaak dat lipopolisakkaried (LPS) vrygestel word in die sirkulasie wat dan 'n kroniese pro-inflammatoriese sitokienrespons dryf. Tans is daar talle studies wat toon dat Aspalathus linearis (rooibos) en sy polifenool aspalatien teen die ontwikkeling van metaboliese siektes beskerm. Die doel van hierdie studie was drievoudig. Eerstens om die effek van LPS op skeletspiergroei en metabolisme te bestudeer deur muis C2C12 mioblaste te gebruik. Tweedens, om die effek(te) wat 3T3-L1 adiposiete op LPS-geïnduseerde pro-inflammatoriese sitokienafskeiding het deur mioblaste te bestudeer, en derdens om te bepaal of 'n aspalatienryke groen rooibosekstrak (Afriplex GRT ™) die LPS-geïnduseerde immuunresponse kan beïnvloed. Om die inflammatoriese skeletspiermodel te vestig, is C2C12 mioblaste 24 uur lank aan LPS (0,1 μg/mL en 1 μg/mL) blootgestel en ook behandel met Afriplex GRTTM (GRT) (1 μg/mL en 10 μg/ml) vir 24 uur. Sellewensvatbaarheid, inflammasie (IL-6-afskeiding), glukose-opname en uitdrukking van relevante gene en proteïene is bepaal. Verder is C2C12 mioblaste gedifferensieer in die teenwoordigheid van LPS om die effekte van inflammasie op miogenese te bepaal. 'n Kokultuurmodel van C2C12 mioblaste en 3T3-L1 pre-adiposiete en adiposiete is gebruik om die afskeiding van IL-6 en adiponektien te bestudeer. LPS was 'n kragtige induseerder van pro-inflammatoriese IL-6 sitokienrespons, spesifiek in mioblaste in teenstelling met miotubules, wat daarop dui dat IL-6 differensiaal in miotubules gereguleer is. As mioblaste tydens differensiasie aan LPS blootgestel word, is daar 'n afname in miotubule-breedte en -getal wat daarop dui dat LPS die spiermassa beïnvloed en moontlik die energiemetabolisme van die skeletspier beïnvloed. Die regulatoriese miogene, miogenien, MioD en miostatien, word deur LPS gedurende die mioblastdifferensiasie afgereguleer. In ‘n ko-kultuur model het LPS die IL-6-afskeiding in beide mioblaste en 3T3-L1-preadiposiete beduidend verhoog, terwyl IL-6-afskeiding deur die gedifferensieerde 3T3-L1-adiposiete in ko-kultuur gemoduleer is. 'n Dramatiese toename in adiponektienvlakke wat deur die gedifferensieerde 3T3- L1-adiposiete afgeskei word, in vergelyking met die pre-adiposiete, het waarskynlik IL-6- afskeiding in die mede-kultuur gemoduleer. GRT kon nie die effekte van LPS-geïnduseerde inflammasie verbeter nie.

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