Are early proteomic and metabolic changes induced by long-term sugar-sweetened beverage consumption the key to unlocking the cardio-metabolic pandemic?

Benade, Janina (2017-03)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: INTRODUCTION: Cardio-metabolic diseases (e.g. type 2 diabetes mellitus) are a major cause of mortality worldwide. The incidence of cardio-metabolic diseases continues to increase, especially in low and middle income countries. This “pandemic” is possibly brought about by a fairly universal shift towards a more “Westernized” diet. High sugar consumption - a hallmark of the “Westernized” diet - may play a key role in the onset of cardio-metabolic diseases. Accordingly, our research focus moved towards sugar-sweetened beverages (SSBs) as it is a major source of added dietary sugars. The current study aimed to elucidate underlying mechanisms leading to the development of cardiometabolic diseases by exploiting a novel rat model of long-term SSB intake, and by focusing on the liver as a major metabolic organ. Here we evaluated well-known systemic markers together with hepatic proteome analysis and downstream consequences. METHODS: Male Wistar rats ( 200 g) were gavaged with 3-5.1 mL SSB daily for three and six months, respectively. The two control groups were gavaged with an iso-volumetric amount of water and iso-caloric amount of butter, respectively. Body weight and systemic blood markers were measured. A proteomic expression analysis was performed on the six-month liver samples. The rest of our experimental work was guided by the proteomic results. Four markers for oxidative stress were evaluated: malondialdehyde, conjugated dienes, reduced:oxidized glutathioneand oxygen radical absorbance capacity. The non-oxidative glucose pathways (NOGPs): polyol pathway, hexosamine biosynthetic pathway, advanced glycation end-products formation and protein kinase C activation; were measured as elevated activity could be indicative of impaired glycolytic flux. The liver histology was investigated with Hematoxylin and Eosin and Masson’s Trichrome stains, respectively. Finally, Western blotting techniques were used to evaluate markers of inflammation. RESULTS: SSB consumption had little effect on systemic markers of cardio-metabolic health. Our proteomic analysis revealed that the expression level of 140 proteins was significantly altered in the SSB group, with a major finding that SSB consumption induces hepatic endoplasmic reticulum (ER) stress. Initially the liver adapted to SSB-mediated nutrient overload by increasing oxidative phosphorylation, suppressing protein transcription, degrading misfolded proteins and improving protein folding capacity. However, due to prolonged stress liver cells entered an ‘’alarm phase’’ marked by a decrease in mitocholdrial metabolism. The proteomic results further revealed that SSB-induced effects are largely attributed to excess caloric intake versus SSBs per se. Surprisingly, oxidative stress did not precede ER stress as there were no significant changes in any of the oxidative stress markers here evaluated. The activity of the NOGPs did not increase significantly thus suggesting that moderate SSB intake did not suppress glucose metabolism and the glycolytic pathway in particular. Conversely, SSB intake increased hepatic lipid storage while limited changes were detected between the groups regarding inflammation and stress signaling. CONCLUSION: Frequent SSB consumption triggers metabolic changes in the liver, i.e. ER stress despite the lack of obvious manifestation of macroscopic “warning signs”. Thus the current study identifies hepatic ER stress as a relatively early result of long-term SSB consumption and it therefore emerges as a unique therapeutic target.

AFRIKAANSE OPSOMMING: INLEIDING: Kardiometaboliese siektes (bv. tipe 2 diabetes mellitus) is wêreldwyd ‘n hoofoorsaak van mortaliteit. Die voorkoms van kardiometaboliese siektes neem voordurend toe, veral in lae en middel-inkomste lande. Hierdie “pandemie” word moontlik gedryf deur ‘n redelike universiële skuif na ‘n meer “Westerse” dieet. Hoë suikerinname, ‘n kerneienskappe van die “Westerse” dieet, speel moontlik ‘n sleutelrol in die ontwikkeling van kardiometaboliese siektes. Daarvolgens fokus ons navorsing op suiker-versoete drankies (SVDs), die hoofbron van ekstra suikers in die dieet. Hierdie studie poog om insig te kry in die onderliggende meganismes wat die ontwikkeling van kardiometaboliese siekes dryf, deur ‘n nuwe rot model vir lang-termyn SVD inname te gebruik en op die lewer as hoof metaboliese orgaan te fokus. Sistemiese merkers, veranderings in die hepatiese proteoom en die nagevolge daarvan is tydens die studie ondersoek. METODES: Manlike Wistar rotte ( 200 g) is daagliks gevoer met 3-5.1 mL SVD deur orale toediening vir drie en ses maande, onderskeidelik. Die kontrole groepe het onderskeidelik ‘n isovolumetriese hoeveelheid water ‘n isokaloriese hoeveelheid botter ontvang. Liggaamsgewig en sistemiese bloedmerkers is gemeet. ‘n Proteomiese uitdrukkinganalise is op die ses maand lewerweefsel uitgevoer. Die res van ons eksperimentele werk is deur die proteomika resultate gerig. Merkers vir oksidatiewe stres is geëvauleer (malonielaldehied, gekonjugeerde diëne, gereduseer tot geoksideerde glutatioonen suurstofradikaal absorbansie kapasiteit). Die nieoksidatiewe glukosepaaie (poliolweg, heksosamien-biosinteseweg, vorming van gevorderde glukeringseindprodukte en proteïen kinase C aktivering) is geëvalueer aangesien verhoogde aktiwiteit ‘n teken van onderdrukte glikolise kan wees. Die lewerhistologie is ondersoek deur “Hematoksilien en Eosien” en “Massons Trichroom” kleuringstegnieke. Westerse blottegnieke is gebruik om merkers van inflammasie te evalueer. RESULTATE: Matige SVD inname het min effek op sistemiese merkers vir kardiometaboliese gesondheid gehad. Die proteomikaanalise wys dat die uitdrukking van 140 proteïene beduidend verander het in die SVD groep en dat SVD inname hepatiese endoplasmiese retikulum- (ER) stres veroorsaak. Aanvanklik het die lewer aangepas by die SVD-bemiddelde voedingstofoorlading deur oksidatiewe fosforilasie te verhoog, transkripsie te verlaag en proteïenvouing en afbreking te bevorder. Weens langdurige stres het die lewer selle egter ‘n “noodfase” betree, gekenmerk deur ‘n afname in mitokondriese metabolisme. Verder dui die resultate dat die effek van SVDs grootliks aan die oormatige kalorieinname te wyte is, eerder as die SVD self. Dit lyk nie asof oksidatiewe stres die voorafgaande “kondisie” is wat ER-stres geïnisiëer het nie aangesien daar geen beduidende veranderings in oksidatiewe stres merkers waargeneem is nie. Die aktiwiteit van die NOGPs het nie beduidend verhoog nie, dus is glukosemetabolisme en die glikolitiese pad in besonder nie onderdruk nie. SVD inname het wel die stoor van lipiede in die lewer verhoog, maar nie inflammasie en stresseinpaaie geïnisiëer nie. GEVOLGTREKKING: Gereelde inname van SVDs lei tot metaboliese veranderings in die lewer soos ER-stres sonder duidelike manifesasie van klassieke vroeë makroskopiese gevaartekens. Die huidige studie indentifiseer hepatiese ER-stres as ‘n relatiewe vroeë resultaat van langtermyn SVD inname en daarom kom dit na vore as ‘n unieke terapeutiese teiken.

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